RNA interference in dermal and fibrotic indications

ABSTRACT

The present invention relates to RNAi constructs with improved tissue and cellular uptake characteristics and methods of use of these compounds in dermal and fibrotic applications.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 15/099,481, entitled “RNA INTERFERENCE IN DERMAL AND FIBROTIC INDICATIONS”, filed Apr. 14, 2016, which is a continuation of U.S. application Ser. No. 13/636,755, entitled “RNA INTERFERENCE IN DERMAL AND FIBROTIC INDICATIONS”, filed Apr. 4, 2013, which is a National Stage Application of PCT/US2011/029867, entitled “RNA INTERFERENCE IN DERMAL AND FIBROTIC INDICATIONS,” filed on Mar. 24, 2011, which was published under PCT Article 21(2) in English and which claims the benefit under 35 U.S.C. § 119(e) of U.S. 61/317,252, entitled “RNA INTERFERENCE IN SKIN INDICATIONS,” filed on Mar. 24, 2010, and U.S. Provisional Application Ser. No. 61/317,633, entitled “RNA INTERFERENCE IN SKIN INDICATIONS,” filed on Mar. 25, 2010, each of which is herein incorporated by reference in its entirety.

FIELD OF INVENTION

The invention pertains to the field of RNA interference (RNAi). The invention more specifically relates to nucleic acid molecules with improved in vivo delivery properties and their use for dermal and fibrotic indications.

BACKGROUND OF INVENTION

Complementary oligonucleotide sequences are promising therapeutic agents and useful research tools in elucidating gene functions. However, prior art oligonucleotide molecules suffer from several problems that may impede their clinical development, and frequently make it difficult to achieve intended efficient inhibition of gene expression (including protein synthesis) using such compositions in vivo.

A major problem has been the delivery of these compounds to cells and tissues. Conventional double-stranded RNAi compounds, 19-29 bases long, form a highly negatively-charged rigid helix of approximately 1.5 by 10-15 nm in size. This rod type molecule cannot get through the cell-membrane and as a result has very limited efficacy both in vitro and in vivo. As a result, all conventional RNAi compounds require some kind of a delivery vehicle to promote their tissue distribution and cellular uptake. This is considered to be a major limitation of the RNAi technology.

There have been previous attempts to apply chemical modifications to oligonucleotides to improve their cellular uptake properties. One such modification was the attachment of a cholesterol molecule to the oligonucleotide. A first report on this approach was by Letsinger et al., in 1989. Subsequently, ISIS Pharmaceuticals, Inc. (Carlsbad, Calif.) reported on more advanced techniques in attaching the cholesterol molecule to the oligonucleotide (Manoharan, 1992).

With the discovery of siRNAs in the late nineties, similar types of modifications were attempted on these molecules to enhance their delivery profiles. Cholesterol molecules conjugated to slightly modified (Soutschek, 2004) and heavily modified (Wolfrum, 2007) siRNAs appeared in the literature. Yamada et al., 2008 also reported on the use of advanced linker chemistries which further improved cholesterol mediated uptake of siRNAs. In spite of all this effort, the uptake of these types of compounds appears to be inhibited in the presence of biological fluids resulting in highly limited efficacy in gene silencing in vivo, limiting the applicability of these compounds in a clinical setting.

SUMMARY OF INVENTION

Described herein is the efficient in vivo delivery of sd-rxRNA molecules to the skin and the use of such molecules for gene silencing. This class of RNAi molecules has superior efficacy both in vitro and in vivo than previously described RNAi molecules. Molecules associated with the invention have widespread potential as therapeutics for disorders or conditions associated with compromised skin and fibrosis.

Aspects of the invention relate to double-stranded ribonucleic acids (dsRNAs) including a sense strand and an antisense strand wherein the antisense strand is complementary to at least 12 contiguous nucleotides of a sequence selected from the sequences within Tables 2, 5, 6, 9, 11, 12, 13, 14, 15, 16, 17 and 23, and wherein the dsRNA is an sd-rxRNA.

Further aspects of the invention relate to double-stranded ribonucleic acids (dsRNAs) comprising a sense strand and an antisense strand wherein the sense strand and/or the antisense strand comprises at least 12 contiguous nucleotides of a sequence selected from the sequences within Tables 1-27, and wherein the dsRNA is an sd-rxRNA.

Further aspects of the invention relate to double-stranded ribonucleic acids (dsRNAs) comprising a sense strand and an antisense strand wherein the antisense strand is complementary to at least 12 contiguous nucleotides of a sequence selected from the sequences within Tables 2, 5, 6, 9, 11, 12, 13, 14, 15, 16, 17 and 23, and wherein the dsRNA is an rxRNAori.

Further aspects of the invention relate to double-stranded ribonucleic acids (dsRNAs) comprising a sense strand and an antisense strand wherein the sense strand and/or the antisense strand comprises at least 12 contiguous nucleotides of a sequence selected from the sequences within Tables 1-27, and wherein the dsRNA is an rxRNAori.

In some embodiments, the dsRNA is directed against CTGF. In some embodiments, the antisense strand of the dsRNA is complementary to at least 12 contiguous nucleotides of a sequence selected from the sequences within Tables 11, 12 and 15. In some embodiments, the sense strand and/or the antisense strand comprises at least 12 contiguous nucleotides of a sequence selected from the sequences within Tables 10, 11, 12, 15, 20 and 24.

In some embodiments, the sense strand comprises at least 12 contiguous nucleotides of a sequence selected from the group consisting of: SEQ ID NOs: 2463, 3429, 2443, 3445, 2459, 3493, 2465 and 3469. In some embodiments, the antisense strand comprises at least 12 contiguous nucleotides of a sequence selected from the group consisting of: 2464, 3430, 4203, 3446, 2460, 3494, 2466 and 3470.

In certain embodiments, the sense strand comprises SEQ ID NO:2463 and the antisense strand comprises SEQ ID NO:2464. In certain embodiments, the sense strand comprises SEQ ID NO:3429 and the antisense strand comprises SEQ ID NO:3430.

In certain embodiments, the sense strand comprises SEQ ID NO:2443 and the antisense strand comprises SEQ ID NO:4203. In certain embodiments, the sense strand comprises SEQ ID NO:3445 and the antisense strand comprises SEQ ID NO:3446.

In certain embodiments, the sense strand comprises SEQ ID NO:2459 and the antisense strand comprises SEQ ID NO:2460. In certain embodiments, the sense strand comprises SEQ ID NO:3493 and the antisense strand comprises SEQ ID NO:3494.

In certain embodiments, the sense strand comprises SEQ ID NO:2465 and the antisense strand comprises SEQ ID NO:2466. In certain embodiments, the sense strand comprises SEQ ID NO:3469 and the antisense strand comprises SEQ ID NO:3470.

In some embodiments, the sense strand comprises at least 12 contiguous nucleotides of a sequence selected from the group consisting of: SEQ ID NOs: 1835, 1847, 1848 and 1849. In certain embodiments, the sense strand comprises a sequence selected from the group consisting of: SEQ ID NOs: 1835, 1847, 1848 and 1849.

In some embodiments, the dsRNA is hydrophobically modified. In certain embodiments, the dsRNA is linked to a hydrophobic conjugate.

Aspects of the invention relate to compositions comprising the dsRNA described herein. In some embodiments, the composition comprises dsRNA directed against genes encoding for more than one protein.

In some embodiments, the composition is formulated for delivery to the skin. In certain embodiments, the composition is in a neutral formulation. In some embodiments, the composition is formulated for topical delivery or for intradermal injection.

Aspects of the invention relate to methods comprising delivering any of the dsRNA described herein or a composition comprising any of the dsRNA described herein to the skin of a subject in need thereof.

Aspects of the invention relate to methods comprising administering to a subject in need thereof a therapeutically effective amount of a double stranded ribonucleic acid (dsRNA) comprising a sense strand and an antisense strand wherein the antisense strand is complementary to at least 12 contiguous nucleotides of a sequence selected from the sequences within Tables 2, 5, 6, 9, 11, 12, 13, 14, 15, 16, 17 and 23, and wherein the dsRNA is an sd-rxRNA.

Further aspects of the invention relate to methods comprising administering to a subject in need thereof a therapeutically effective amount of a double stranded ribonucleic acid (dsRNA) comprising a sense strand and an antisense strand wherein the sense strand and/or the antisense strand comprises at least 12 contiguous nucleotides of a sequence selected from the sequences within Tables 1-27, and wherein the dsRNA is an sd-rxRNA.

Further aspects of the invention relate to methods comprising administering to a subject in need thereof a therapeutically effective amount of a double stranded ribonucleic acid (dsRNA) comprising a sense strand and an antisense strand wherein the antisense strand is complementary to at least 12 contiguous nucleotides of a sequence selected from the sequences within Tables 2, 5, 6, 9, 11, 12, 13, 14, 15, 16, 17 and 23, and wherein the dsRNA is an rxRNAori.

Further aspects of the invention relate to methods comprising administering to a subject in need thereof a therapeutically effective amount of a double stranded ribonucleic acid (dsRNA) comprising a sense strand and an antisense strand wherein the sense strand and/or the antisense strand comprises at least 12 contiguous nucleotides of a sequence selected from the sequences within Tables 1-27, and wherein the dsRNA is an rxRNAori.

In some embodiments, the method is a method for treating compromised skin. In some embodiments, the method is a method for treating or preventing a fibrotic disorder.

In some embodiments, the dsRNA is administered via intradermal injection. In some embodiments, the dsRNA is administered locally to the skin. In some embodiments, two or more nucleic acid molecules are administered simultaneously or sequentially.

In some embodiments, one or more of the dsRNAs is hydrophobically modified. In certain embodiments, one or more of the dsRNAs is linked to a hydrophobic conjugate.

In some embodiments, the dsRNA is directed against CTGF. In certain embodiments, the antisense strand of the dsRNA is complementary to at least 12 contiguous nucleotides of a sequence selected from the sequences within Tables 11, 12 and 15. In some embodiments, the sense strand and/or the antisense strand comprises at least 12 contiguous nucleotides of a sequence selected from the sequences within Tables 10, 11, 12, 15, 20 and 24.

In some embodiments, the sense strand comprises at least 12 contiguous nucleotides of a sequence selected from the group consisting of: SEQ ID NOs: 2463, 3429, 2443, 3445, 2459, 3493, 2465 and 3469. In certain embodiments, the antisense strand comprises at least 12 contiguous nucleotides of a sequence selected from the group consisting of: 2464, 3430, 4203, 3446, 2460, 3494, 2466 and 3470.

In certain embodiments, the sense strand comprises SEQ ID NO:2463 and the antisense strand comprises SEQ ID NO:2464. In certain embodiments, the sense strand comprises SEQ ID NO:3429 and the antisense strand comprises SEQ ID NO:3430.

In certain embodiments, the sense strand comprises SEQ ID NO:2443 and the antisense strand comprises SEQ ID NO:4203. In certain embodiments, the sense strand comprises SEQ ID NO:3445 and the antisense strand comprises SEQ ID NO:3446.

In certain embodiments, the sense strand comprises SEQ ID NO:2459 and the antisense strand comprises SEQ ID NO:2460. In certain embodiments, the sense strand comprises SEQ ID NO:3493 and the antisense strand comprises SEQ ID NO:3494.

In certain embodiments, the sense strand comprises SEQ ID NO:2465 and the antisense strand comprises SEQ ID NO:2466. In certain embodiments, the sense strand comprises SEQ ID NO:3469 and the antisense strand comprises SEQ ID NO:3470.

In some embodiments, the sense strand comprises at least 12 contiguous nucleotides of a sequence selected from the group consisting of: SEQ ID NOs: 1835, 1847, 1848 and 1849. In some embodiments, the sense strand comprises a sequence selected from the group consisting of: SEQ ID NOs: 1835, 1847, 1848 and 1849.

Aspects of the invention relate to treating or preventing a fibrotic disorder. In some embodiments, the fibrotic disorder is selected from the group consisting of pulmonary fibrosis, liver cirrhosis, scleroderma and glomerulonephritis, lung fibrosis, liver fibrosis, skin fibrosis, muscle fibrosis, radiation fibrosis, kidney fibrosis, proliferative vitreoretinopathy, restenosis and uterine fibrosis, and trabeculectomy failure due to scarring.

In some embodiments, the dsRNA are administered via intradermal injection, while in other embodiments, the one or more dsRNA are administered subcutaneously or epicutaneously.

The one or more dsRNA can be administered prior to, during and/or after a medical procedure. In some embodiments, administration occurs within 8 days prior to or within 8 days after the medical procedure. In some embodiments, the medical procedure is surgery. In certain embodiments, the surgery is elective. In some embodiments, the surgery comprises epithelial grafting or skin grafting. In some embodiments, the one or more double stranded nucleic acid molecules are administered to a graft donor site and/or a graft recipient site.

Aspects of the invention relate to methods for administering one or more dsRNA prior to, during and/or after an injury. In some embodiments, the subject has a wound such as a chronic wound. In certain embodiments, the wound is a result of elective surgery. The wound can be external or internal. In some embodiments, the dsRNA is administered after burn injury.

Methods described herein include methods for promoting wound healing and methods for preventing scarring.

In some embodiments, one or more of the dsRNA administered to a subject is directed against a gene selected from the group consisting of TGFB1, TGFB2, hTGFB1, hTGFB2, PTGS2, SPP1, hSPP1, CTGF or hCTGF. In some embodiments, the one or more dsRNA are administered on the skin of the subject. In certain embodiments, the one or more dsRNA molecules are in the form of a cream or ointment. In some embodiments, two or more or three or more nucleic acids are administered. Two or more nucleic acid molecules can be administered simultaneously or sequentially.

Aspects of the invention related to nucleic acids that are optimized. In some embodiments, one or more double stranded nucleic acid molecules are hydrophobically modified. In certain embodiments, the one or more double stranded nucleic acid molecules are linked to a hydrophobic conjugate or multiple hydrophobic conjugates. In some embodiments, the one or more double stranded nucleic acid molecule are linked to a lipophilic group. In certain embodiments, the lipophilic group is linked to the passenger strand of the one or more double stranded nucleic acid molecules. In some embodiment, the one or more double stranded nucleic acid molecules are linked to cholesterol, a long chain alkyl cholesterol analog, vitamin A or vitamin E. In some embodiments, the one or more double stranded nucleic acid molecules is attached to chloroformate.

Aspects of the invention related to nucleic acids that are optimized through modifications. In some embodiments, the one or more double stranded nucleic acid molecules includes at least one 2′ O methyl or 2′ fluoro modification and/or at least one methyl C or U modification. In some embodiments, the one or more double stranded nucleic acid molecules has a guide strand of 16-28 nucleotides in length. In certain embodiments, at least 40% of the nucleotides of the one or more double stranded nucleic acid molecules are modified. Double stranded nucleic acid molecules described herein can also be attached to linkers. In some embodiments, the linker is protonatable.

Aspects of the invention relate to double stranded nucleic acid molecules that contain at least two single stranded regions. In some embodiments, the single stranded regions contain phosphorothioate modifications. In certain embodiments, the single stranded regions are located at the 3′ end of the guide strand and the 5′ end of the passenger strand.

Aspects of the invention relate to methods for delivering a nucleic acid to a subject, involving administering to a subject within 8 days prior to a medical procedure a therapeutically effective amount for treating compromised skin of one or more sd-rxRNAs.

Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention. This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures is represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:

FIG. 1 demonstrates the expression profiles for non-limiting examples of target genes including MAP4K4, SPP1, CTGF, PTGS2 and TGFB1. As expected, target gene expression is elevated early and returns to normal by day 10.

FIG. 2 presents schematics depicting an experimental approach to visualizing tissue after intradermal injection.

FIG. 3 demonstrates silencing of MAP4K4 following intradermal injection of sd-rxRNA targeting MAP4K4.

FIG. 4 demonstrates silencing of MAP4K4, PPIB and CTGF following intradermal injection of sd-rxRNA molecules targeting each gene.

FIG. 5 demonstrates silencing of MAP4K4 following intradermal injection of sd-rxRNA targeting MAP4K4. Normalized expression of MAP4K4 relative to controls is demonstrated.

FIG. 6 demonstrates silencing of PPIB following intradermal injection of sd-rxRNA targeting PPIB. Normalized expression of PPIB relative to controls is demonstrated.

FIG. 7 demonstrates the duration of PPIB silencing following intradermal injection of sd-rxRNA targeting PPIB.

FIG. 8 demonstrates the duration of MAP4K4 silencing following intradermal injection of sd-rxRNA targeting MAP4K4.

FIG. 9 demonstrates equivalent silencing achieved using two different dosing regimens.

FIG. 10 demonstrates examples of sd-rxRNA molecules targeting CTGF that are efficacious for gene silencing.

FIG. 11 demonstrates examples of sd-rxRNA molecules targeting CTGF that are efficacious for gene silencing.

FIG. 12 demonstrates a dose response for sd-rxRNA molecules targeting CTGF.

FIG. 13 demonstrates a sample of an original sd-rxRNA screen.

FIG. 14 presents data on a hit from the original sd-rxRNA screen.

FIG. 15 demonstrates gene expression of PTGS2 following administration of sd-rxRNA targeting PTGS2.

FIG. 16 demonstrates gene expression of hTGFB1 following administration of sd-rxRNA targeting hTGFB1.

FIG. 17 demonstrates gene expression of hTGFB1 following administration of sd-rxRNA targeting hTGFB1.

FIG. 18 demonstrates results of TGFB1 sd-rxRNA screening.

FIG. 19 demonstrates gene expression of TGFB2 following administration of sd-rxRNA targeting TGFB2.

FIG. 20 demonstrates gene expression of TGFB2 following administration of sd-rxRNA targeting TGFB2.

FIG. 21 demonstrates gene expression of TGFB2 following administration of sd-rxRNA targeting TGFB2.

FIG. 22 demonstrates gene expression of TGFB2 following administration of sd-rxRNA targeting TGFB2.

FIG. 23 demonstrates gene expression of TGFB2 following administration of sd-rxRNA targeting TGFB2.

FIG. 24 demonstrates results of TGFB2 sd-rxRNA screening.

FIG. 25 demonstrates identification of potent hSPP1 sd-rxRNAs.

FIG. 26 demonstrates identification of potent hSPP1 sd-rxRNAs.

FIG. 27 demonstrates identification of potent hSPP1 sd-rxRNAs.

FIG. 28 demonstrates SPP1 sd-rxRNA compound selection.

FIG. 29 demonstrates that variation of linker chemistry does not influence silencing activity of sd-rxRNAs in vitro. Two different linker chemistries were evaluated, a hydroxyproline linker and ribo linker, on multiple sd-rxRNAs (targeting Map4k4 or PPIB) in passive uptake assays to determine linkers which favor self delivery. HeLa cells were transfected in the absence of a delivery vehicle (passive transfection) with sd-rxRNAs at 1 uM, 0.1 uM or 0.01 uM for 48 hrs. Use of either linker results in an efficacious delivery of sd-rxRNA.

FIG. 30 depicts CTGF as a central factor in the pathway to fibrosis.

FIG. 31 depicts the phases of wound healing.

FIG. 32 depicts the chemical optimization of sd-rxRNA leads.

FIG. 33 demonstrates that chemically optimized CTGF L1 sd-rxRNAs are active.

FIG. 34 demonstrates in vitro efficacy of chemically optimized CTGF L1 sd-rxRNAs.

FIG. 35 demonstrates in vitro stability of chemically optimized CTGF L1 sd-rxRNAs.

FIG. 36 demonstrates that chemically optimized CTGF L2 sd-rxRNAs are active.

FIG. 37 demonstrates in vitro efficacy of chemically optimized CTGF L2 sd-rxRNAs.

FIG. 38 demonstrates in vitro stability of chemically optimized CTGF L2 sd-rxRNAs.

FIG. 39 provides a summary of compounds that are active in vivo.

FIG. 40 demonstrates that treatment with CTGF LIB target sequence resulted in mRNA silencing.

FIG. 41 demonstrates that treatment with CTGF L2 target sequence resulted in mRNA silencing.

FIG. 42 demonstrates CTGF silencing after two intradermal injections of RXi-109.

FIG. 43 demonstrates the duration of CTGF silencing in skin after intradermal injection of the sd-rxRNA in SD rats. Eight millimeter skin biopsies were harvested, and mRNA levels were quantified by QPCR and normalized to a housekeeping gene. Shown is percent (%) silencing vs. Non Targeting Control (NTC); PBS at each time point is one experimental group; * p≤0.04; ** p≤0.002.

FIG. 44 demonstrates that chemically optimized CTGF L3 sd-rxRNAs are active.

FIG. 45 demonstrates absolute luminescence of CTGF L4 sd-rxRNAs.

FIG. 46 demonstrates that chemically optimized CTGF L4 sd-rxRNAs are active.

FIG. 47 demonstrates changes in mRNA expression levels of CTGF, α-SM actin, collagen 1A2, and collagen 3A1 after intradermal injection of CTFG sd-rxRNA in SD rats. mRNA levels were quantified by qPCR.

FIG. 48 demonstrates that there is no apparent delay in wound healing with treatment of CTGF-targeting sd-rxRNA. Some changes was observed with treatment of a combination of CTGF- and COX2-targeting sd-rxRNAs.

FIG. 49 demonstrates that administration of sd-rxRNAs decreases wound width over the course of at least 9 days. The graph shows microscopic measurements of wound width in rats on days 3, 6, and 9 post-wounding. Each group represents 5 rats. Two non-serial sections from each wound were measured and the average width of the two was calculated per wound. *p<0.05 vs. PBS an NTC.

FIG. 50 demonstrates that administration of sd-rxRNAs decreases wound area over the course of at least 9 days. The graph shows microscopic measurements of wound width in rats on days 3, 6, and 9 post-wounding. Each group represents 5 rats. Two non-serial sections from each wound were measured and the average width of the two was calculated per wound. *p<0.05 vs. PBS an NTC.

FIG. 51 demonstrates that administration of sd-rxRNAs increase the percentage of wound re-epithelialization over the course of at least 9 days. The graph shows microscopic measurements of wound width in rats on days 3, 6, and 9 post-wounding. Each group represents 5 rats. Two non-serial sections from each wound were measured and the average width of the two was calculated per wound. *p<0.05 vs. PBS an NTC.

FIG. 52 demonstrates that administration of sd-rxRNAs increases the average granulation tissue maturity scores over the course of at least 9 days. The graph shows microscopic measurements of wound width in rats on days 3, 6, and 9 post-wounding (5=mature, 1=immature). Each group represents 5 rats.

FIG. 53 demonstrates CD68 labeling in day 9 wounds (0=no labeling, 3=substantial labeling). Each group represents 5 rats.

FIG. 54 demonstrates that CTGF leads have different toxicity levels in vitro.

FIG. 55 shows percentage (%) of cell viability after RXI 109 dose escalation (oligos formulated in PBS).

FIG. 56 is a schematic of Phases 1 and 2 clinical trial design.

FIG. 57 is a schematic of Phases 1 and 2 clinical trial design.

FIG. 58 demonstrates a percent (%) decrease in PPIB expression in the liver relative to PBS control. Lipoid formulated rxRNAs (10 mg/kg) were delivery systemically to Balb/c mice (n=5) by single tail vein injections. Liver tissue was harvested at 24 hours after injection and expression was analyzed by qPCR (normalized to β-actin). Map4K4 rxRNAori also showed significant silencing (˜83%, p<0.001) although Map4K4 sd-rxRNA did not significantly reduce target gene expression (˜17%, p=0.019). TD.035.2278, Published lipidoid delivery reagent, 98N12-5(1), from Akinc, 2009.

FIG. 59 demonstrates that chemically optimized PTGS2 L1 sd-rxRNAs are active.

FIG. 60 demonstrates that chemically optimized PTGS2 L2 sd-rxRNAs are active.

FIG. 61 demonstrates that chemically optimized hTGFB1 L1 sd-rxRNAs are active.

FIG. 62 demonstrates that chemically optimized hTGFB1 L1 sd-rxRNAs are active.

FIG. 63 demonstrates that chemically optimized hTGFB2 L1 sd-rxRNAs are active.

FIG. 64 demonstrates that chemically optimized hTGFB2 sd-rxRNAs are active.

DETAILED DESCRIPTION

Aspects of the invention relate to methods and compositions involved in gene silencing. The invention is based at least in part on the surprising discovery that administration of sd-rxRNA molecules to the skin, such as through intradermal injection or subcutaneous administration, results in efficient silencing of gene expression in the skin. Highly potent sd-rxRNA molecules that target genes including SPP1, CTGF, PTGS2, TGFB1 and TGFB2 were also identified herein through cell-based screening. sd-rxRNAs represent a new class of therapeutic RNAi molecules with significant potential in treatment of compromised skin.

sd-rxRNA Molecules

Aspects of the invention relate to sd-rxRNA molecules. As used herein, an “sd-rxRNA” or an “sd-rxRNA molecule” refers to a self-delivering RNA molecule such as those described in, and incorporated by reference from, PCT Publication No. WO2010/033247 (Application No. PCT/US2009/005247), filed on Sep. 22, 2009, and entitled “REDUCED SIZE SELF-DELIVERING RNAI COMPOUNDS,” and PCT application PCT/US2009/005246, filed on Sep. 22, 2009, and entitled “RNA INTERFERENCE IN SKIN INDICATIONS.” Briefly, an sd-rxRNA, (also referred to as an sd-rxRNA^(nano)) is an isolated asymmetric double stranded nucleic acid molecule comprising a guide strand, with a minimal length of 16 nucleotides, and a passenger strand of 8-18 nucleotides in length, wherein the double stranded nucleic acid molecule has a double stranded region and a single stranded region, the single stranded region having 4-12 nucleotides in length and having at least three nucleotide backbone modifications. In preferred embodiments, the double stranded nucleic acid molecule has one end that is blunt or includes a one or two nucleotide overhang. sd-rxRNA molecules can be optimized through chemical modification, and in some instances through attachment of hydrophobic conjugates.

In some embodiments, an sd-rxRNA comprises an isolated double stranded nucleic acid molecule comprising a guide strand and a passenger strand, wherein the region of the molecule that is double stranded is from 8-15 nucleotides long, wherein the guide strand contains a single stranded region that is 4-12 nucleotides long, wherein the single stranded region of the guide strand contains 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 phosphorothioate modifications, and wherein at least 40% of the nucleotides of the double stranded nucleic acid are modified.

The polynucleotides of the invention are referred to herein as isolated double stranded or duplex nucleic acids, oligonucleotides or polynucleotides, nano molecules, nano RNA, sd-rxRNA^(nano), sd-rxRNA or RNA molecules of the invention.

sd-rxRNAs are much more effectively taken up by cells compared to conventional siRNAs. These molecules are highly efficient in silencing of target gene expression and offer significant advantages over previously described RNAi molecules including high activity in the presence of serum, efficient self delivery, compatibility with a wide variety of linkers, and reduced presence or complete absence of chemical modifications that are associated with toxicity.

In contrast to single-stranded polynucleotides, duplex polynucleotides have traditionally been difficult to deliver to a cell as they have rigid structures and a large number of negative charges which makes membrane transfer difficult. sd-rxRNAs however, although partially double-stranded, are recognized in vivo as single-stranded and, as such, are capable of efficiently being delivered across cell membranes. As a result the polynucleotides of the invention are capable in many instances of self delivery. Thus, the polynucleotides of the invention may be formulated in a manner similar to conventional RNAi agents or they may be delivered to the cell or subject alone (or with non-delivery type carriers) and allowed to self deliver. In one embodiment of the present invention, self delivering asymmetric double-stranded RNA molecules are provided in which one portion of the molecule resembles a conventional RNA duplex and a second portion of the molecule is single stranded.

The oligonucleotides of the invention in some aspects have a combination of asymmetric structures including a double stranded region and a single stranded region of nucleotides or longer, specific chemical modification patterns and are conjugated to lipophilic or hydrophobic molecules. This class of RNAi like compounds have superior efficacy in vitro and in vivo. It is believed that the reduction in the size of the rigid duplex region in combination with phosphorothioate modifications applied to a single stranded region contribute to the observed superior efficacy.

The invention is based at least in part on the surprising discovery that sd-rxRNA molecules are delivered efficiently in vivo to the skin through a variety of methods including intradermal injection and subcutaneous administration. Furthermore, sd-rxRNA molecules are efficient in mediating gene silencing in the region of the skin where they are targeted.

Aspects of the invention relate to the use of cell-based screening to identify potent sd-rxRNA molecules. Described herein is the identification of potent sd-rxRNA molecules that target a subset of genes including SPP1, CTFG, PTGS2. TGFB1 and TGFB2. In some embodiments, a target gene is selected and an algorithm is applied to identify optimal target sequences within that gene (Example 2). For example, many sequences can be selected for one gene. In some instances, the sequences that are identified are generated as RNAi compounds for a first round of testing. For example, the RNAi compounds based on the optimal predicted sequences can initially be generated as rxRNAori (“ori”) sequences for the first round of screening. After identifying potent RNAi compounds, these can be generated as sd-rxRNA molecules.

dsRNA formulated according to the invention also includes rxRNAori. rxRNAori refers to a class of RNA molecules described in and incorporated by reference from PCT Publication No. WO2009/102427 (Application No. PCT/US2009/000852), filed on Feb. 11, 2009, and entitled, “MODIFIED RNAI POLYNUCLEOTIDES AND USES THEREOF.”

In some embodiments, an rxRNAori molecule comprises a double-stranded RNA (dsRNA) construct of 12-35 nucleotides in length, for inhibiting expression of a target gene, comprising: a sense strand having a 5′-end and a 3′-end, wherein the sense strand is highly modified with 2′-modified ribose sugars, and wherein 3-6 nucleotides in the central portion of the sense strand are not modified with 2′-modified ribose sugars and, an antisense strand having a 5′-end and a 3′-end, which hybridizes to the sense strand and to mRNA of the target gene, wherein the dsRNA inhibits expression of the target gene in a sequence-dependent manner.

rxRNAori can contain any of the modifications described herein. In some embodiments, at least 30% of the nucleotides in the rxRNAori are modified. For example, at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the nucleotides in the rxRNAori are modified. In some embodiments, 100% of the nucleotides in the sd-rxRNA are modified. In some embodiments, only the passenger strand of the rxRNAori contains modifications.

In some embodiments, the RNAi compounds of the invention comprise an asymmetric compound comprising a duplex region (required for efficient RISC entry of 8-15 bases long) and single stranded region of 4-12 nucleotides long; with a 13 or 14 nucleotide duplex. A 6 or 7 nucleotide single stranded region is preferred in some embodiments. The single stranded region of the new RNAi compounds also comprises 2-12 phosphorothioate internucleotide linkages (referred to as phosphorothioate modifications). 6-8 phosphorothioate internucleotide linkages are preferred in some embodiments. Additionally, the RNAi compounds of the invention also include a unique chemical modification pattern, which provides stability and is compatible with RISC entry. The combination of these elements has resulted in unexpected properties which are highly useful for delivery of RNAi reagents in vitro and in vivo.

The chemical modification pattern, which provides stability and is compatible with RISC entry includes modifications to the sense, or passenger, strand as well as the antisense, or guide, strand. For instance the passenger strand can be modified with any chemical entities which confirm stability and do not interfere with activity. Such modifications include 2′ ribo modifications (O-methyl, 2′ F, 2 deoxy and others) and backbone modification like phosphorothioate modifications. A preferred chemical modification pattern in the passenger strand includes Omethyl modification of C and U nucleotides within the passenger strand or alternatively the passenger strand may be completely Omethyl modified.

The guide strand, for example, may also be modified by any chemical modification which confirms stability without interfering with RISC entry. A preferred chemical modification pattern in the guide strand includes the majority of C and U nucleotides being 2′ F modified and the 5′ end being phosphorylated. Another preferred chemical modification pattern in the guide strand includes 2′Omethyl modification of position 1 and C/U in positions 11-18 and 5′ end chemical phosphorylation. Yet another preferred chemical modification pattern in the guide strand includes 2′Omethyl modification of position 1 and C/U in positions 11-18 and 5′ end chemical phosphorylation and 2′F modification of C/U in positions 2-10. In some embodiments the passenger strand and/or the guide strand contains at least one 5-methyl C or U modifications.

In some embodiments, at least 30% of the nucleotides in the sd-rxRNA are modified. For example, at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the nucleotides in the sd-rxRNA are modified. In some embodiments, 100% of the nucleotides in the sd-rxRNA are modified.

The above-described chemical modification patterns of the oligonucleotides of the invention are well tolerated and actually improved efficacy of asymmetric RNAi compounds.

It was also demonstrated experimentally herein that the combination of modifications to RNAi when used together in a polynucleotide results in the achievement of optimal efficacy in passive uptake of the RNAi. Elimination of any of the described components (Guide strand stabilization, phosphorothioate stretch, sense strand stabilization and hydrophobic conjugate) or increase in size in some instances results in sub-optimal efficacy and in some instances complete lost of efficacy. The combination of elements results in development of a compound, which is fully active following passive delivery to cells such as HeLa cells.

The data in the Examples presented below demonstrates high efficacy of the oligonucleotides of the invention both in vitro in variety of cell types and in vivo upon local and systemic administration.

The sd-rxRNA can be further improved in some instances by improving the hydrophobicity of compounds using of novel types of chemistries. For example one chemistry is related to use of hydrophobic base modifications. Any base in any position might be modified, as long as modification results in an increase of the partition coefficient of the base. The preferred locations for modification chemistries are positions 4 and 5 of the pyrimidines. The major advantage of these positions is (a) ease of synthesis and (b) lack of interference with base-pairing and A form helix formation, which are essential for RISC complex loading and target recognition. A version of sd-rxRNA compounds where multiple deoxy Uridines are present without interfering with overall compound efficacy was used. In addition major improvement in tissue distribution and cellular uptake might be obtained by optimizing the structure of the hydrophobic conjugate. In some of the preferred embodiment the structure of sterol is modified to alter (increase/decrease) C17 attached chain. This type of modification results in significant increase in cellular uptake and improvement of tissue uptake prosperities in vivo.

Aspects of the invention relate to double-stranded ribonucleic acid molecules (dsRNA) such as sd-rxRNA and rxRNAori. dsRNA associated with the invention can comprise a sense strand and an antisense strand wherein the antisense strand is complementary to at least 12 contiguous nucleotides of a sequence selected from the sequences within Tables 2, 5, 6, 9, 11, 12, 13, 14, 15, 16, 17 and 23. For example, the antisense strand can be complementary to at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 contiguous nucleotides, or can be complementary to 25 nucleotides of a sequence selected from the sequences within Tables 2, 5, 6, 9, 11, 12, 13, 14, 15, 16, 17 and 23.

dsRNA associated with the invention can comprise a sense strand and an antisense strand wherein the sense strand and/or the antisense strand comprises at least 12 contiguous nucleotides of a sequence selected from the sequences within Tables 1-27. For example, the sense strand and/or the antisense strand can comprise at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 contiguous nucleotides, or can comprise 25 nucleotides of a sequence selected from the sequences within Tables 1-27.

Aspects of the invention relate to dsRNA directed against CTGF. For example, the antisense strand of a dsRNA directed against CTGF can be complementary to at least 12 contiguous nucleotides of a sequence selected from the sequences within Tables 11, 12 and 15. The sense strand and/or the antisense strand of a dsRNA directed against CTGF can comprises at least 12 contiguous nucleotides of a sequence selected from the sequences within Tables 10, 11, 12, 15, 20 and 24.

In some embodiments, the sense strand comprises at least 12 contiguous nucleotides of a sequence selected from the group consisting of: SEQ ID NOs: 2463, 3429, 2443, 3445, 2459, 3493, 2465 and 3469. In certain embodiments, the sense strand comprises or consists of a sequence selected from the group consisting of: SEQ ID NOs: 2463, 3429, 2443, 3445, 2459, 3493, 2465 and 3469.

In some embodiments, the antisense strand comprises at least 12 contiguous nucleotides of a sequence selected from the group consisting of: 2464, 3430, 4203, 3446, 2460, 3494, 2466 and 3470. In certain embodiments, the antisense strand comprises or consists of a sequence selected from the group consisting of: 2464, 3430, 4203, 3446, 2460, 3494, 2466 and 3470.

In a preferred embodiment, the sense strand comprises SEQ ID NO:2463 (GCACCUUUCUAGA) and the antisense strand comprises SEQ ID NO:2464 (UCUAGAAAGGUGCAAACAU). The sequences of SEQ ID NO:2463 and SEQ ID NO:2464 can be modified in a variety of ways according to modifications described herein. A preferred modification pattern for SEQ ID NO:2463 is depicted by SEQ ID NO:3429 (G.mC. A.mC.mC.mU.mU.mU.mC.mU. A*mG*mA.TEG-Chl). A preferred modification pattern for SEQ ID NO:2464 is depicted by SEQ ID NO:3430 (P.mU.fC.fU. A. G.mA. A.mA. G. G.fU. G.mC* A* A* A*mC* A* U). An sd-rxRNA consisting of SEQ ID NO:3429 and SEQ ID NO:3430 is also referred to as RXi-109.

In another preferred embodiment, the sense strand comprises SEQ ID NO:2443 (UUGCACCUUUCUAA) and the antisense strand comprises SEQ ID NO:4203 (UUAGAAAGGUGCAAACAAGG). The sequences of SEQ ID NO:2443 and SEQ ID NO:4203 can be modified in a variety of ways according to modifications described herein. A preferred modification pattern for SEQ ID NO:2443 is depicted by SEQ ID NO:3445 (mU.mU. G.mC. A.mC.mC.mU.mU.mU.mC.mU*mA*mA.TEG-Chl). A preferred modification pattern for SEQ ID NO:4203 is depicted by SEQ ID NO:3446 (P.mU.fU. A. G. A.mA. A. G. G.fU. G.fC.mA.mA*mA*fC*mA*mA*mG* G.).

In another preferred embodiment, the sense strand comprises SEQ ID NO:2459 (GUGACCAAAAGUA) and the antisense strand comprises SEQ ID NO:2460 (UACUUUUGGUCACACUCUC). The sequences of SEQ ID NO:2459 and SEQ ID NO:2460 can be modified in a variety of ways according to modifications described herein. A preferred modification pattern for SEQ ID NO:2459 is depicted by SEQ ID NO:3493 (G.mU. G. A.mC.mC. A. A. A. A. G*mU*mA.TEG-Chl). A preferred modification pattern for SEQ ID NO:2460 is depicted by SEQ ID NO:3494 (P.mU. A.fC.fU.fU.fU.fU. G. G.fU.mC. A.mC* A*mC*mU*mC*mU* C.).

In another preferred embodiment, the sense strand comprises SEQ ID NO:2465 (CCUUUCUAGUUGA) and the antisense strand comprises SEQ ID NO:2466 (UCAACUAGAAAGGUGCAAA). The sequences of SEQ ID NO:2465 and SEQ ID NO:2466 can be modified in a variety of ways according to modifications described herein. A preferred modification pattern for SEQ ID NO:2465 is depicted by SEQ ID NO:3469 (mC.mC.mU.mU.mU.mC.mU. A. G.mU.mU*mG*mA.TEG-Chl). A preferred modification pattern for SEQ ID NO:2466 is depicted by SEQ ID NO:3470 (P.mU.fC. A. A.fC.fU. A. G. A.mA. A. G. G*fU*mG*fC*mA*mA* A.).

A preferred embodiment of an rxRNAori directed against CTGF can comprise at least 12 contiguous nucleotides of a sequence selected from the group consisting of: SEQ ID NOs:1835, 1847, 1848 and 1849. In some embodiments, the sense strand of the rxRNAori comprises or consists of SEQ ID NOs:1835, 1847, 1848 or 1849.

Aspects of the invention relate to compositions comprising dsRNA such as sd-rxRNA and rxRNAori. In some embodiments compositions comprise two or more dsRNA that are directed against different genes.

This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having,” “containing,” “involving,” and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

Thus, aspects of the invention relate to isolated double stranded nucleic acid molecules comprising a guide (antisense) strand and a passenger (sense) strand. As used herein, the term “double-stranded” refers to one or more nucleic acid molecules in which at least a portion of the nucleomonomers are complementary and hydrogen bond to form a double-stranded region. In some embodiments, the length of the guide strand ranges from 16-29 nucleotides long. In certain embodiments, the guide strand is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 nucleotides long. The guide strand has complementarity to a target gene. Complementarity between the guide strand and the target gene may exist over any portion of the guide strand. Complementarity as used herein may be perfect complementarity or less than perfect complementarity as long as the guide strand is sufficiently complementary to the target that it mediates RNAi. In some embodiments complementarity refers to less than 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% mismatch between the guide strand and the target. Perfect complementarity refers to 100% complementarity. Thus the invention has the advantage of being able to tolerate sequence variations that might be expected due to genetic mutation, strain polymorphism, or evolutionary divergence. For example, siRNA sequences with insertions, deletions, and single point mutations relative to the target sequence have also been found to be effective for inhibition. Moreover, not all positions of a siRNA contribute equally to target recognition. Mismatches in the center of the siRNA are most critical and essentially abolish target RNA cleavage. Mismatches upstream of the center or upstream of the cleavage site referencing the antisense strand are tolerated but significantly reduce target RNA cleavage. Mismatches downstream of the center or cleavage site referencing the antisense strand, preferably located near the 3′ end of the antisense strand, e.g. 1, 2, 3, 4, 5 or 6 nucleotides from the 3′ end of the antisense strand, are tolerated and reduce target RNA cleavage only slightly.

While not wishing to be bound by any particular theory, in some embodiments, the guide strand is at least 16 nucleotides in length and anchors the Argonaute protein in RISC. In some embodiments, when the guide strand loads into RISC it has a defined seed region and target mRNA cleavage takes place across from position 10-11 of the guide strand. In some embodiments, the 5′ end of the guide strand is or is able to be phosphorylated. The nucleic acid molecules described herein may be referred to as minimum trigger RNA.

In some embodiments, the length of the passenger strand ranges from 8-15 nucleotides long. In certain embodiments, the passenger strand is 8, 9, 10, 11, 12, 13, 14 or 15 nucleotides long. The passenger strand has complementarity to the guide strand. Complementarity between the passenger strand and the guide strand can exist over any portion of the passenger or guide strand. In some embodiments, there is 100% complementarity between the guide and passenger strands within the double stranded region of the molecule.

Aspects of the invention relate to double stranded nucleic acid molecules with minimal double stranded regions. In some embodiments the region of the molecule that is double stranded ranges from 8-15 nucleotides long. In certain embodiments, the region of the molecule that is double stranded is 8, 9, 10, 11, 12, 13, 14 or 15 nucleotides long. In certain embodiments the double stranded region is 13 or 14 nucleotides long. There can be 100% complementarity between the guide and passenger strands, or there may be one or more mismatches between the guide and passenger strands. In some embodiments, on one end of the double stranded molecule, the molecule is either blunt-ended or has a one-nucleotide overhang. The single stranded region of the molecule is in some embodiments between 4-12 nucleotides long. For example the single stranded region can be 4, 5, 6, 7, 8, 9, 10, 11 or 12 nucleotides long. However, in certain embodiments, the single stranded region can also be less than 4 or greater than 12 nucleotides long. In certain embodiments, the single stranded region is 6 nucleotides long.

RNAi constructs associated with the invention can have a thermodynamic stability (ΔG) of less than −13 kkal/mol. In some embodiments, the thermodynamic stability (ΔG) is less than −20 kkal/mol. In some embodiments there is a loss of efficacy when (ΔG) goes below −21 kkal/mol. In some embodiments a (ΔG) value higher than −13 kkal/mol is compatible with aspects of the invention. Without wishing to be bound by any theory, in some embodiments a molecule with a relatively higher (ΔG) value may become active at a relatively higher concentration, while a molecule with a relatively lower (ΔG) value may become active at a relatively lower concentration. In some embodiments, the (ΔG) value may be higher than −9 kkcal/mol. The gene silencing effects mediated by the RNAi constructs associated with the invention, containing minimal double stranded regions, are unexpected because molecules of almost identical design but lower thermodynamic stability have been demonstrated to be inactive (Rana et al. 2004).

Without wishing to be bound by any theory, results described herein suggest that a stretch of 8-10 bp of dsRNA or dsDNA will be structurally recognized by protein components of RISC or co-factors of RISC. Additionally, there is a free energy requirement for the triggering compound that it may be either sensed by the protein components and/or stable enough to interact with such components so that it may be loaded into the Argonaute protein. If optimal thermodynamics are present and there is a double stranded portion that is preferably at least 8 nucleotides then the duplex will be recognized and loaded into the RNAi machinery.

In some embodiments, thermodynamic stability is increased through the use of LNA bases. In some embodiments, additional chemical modifications are introduced. Several non-limiting examples of chemical modifications include: 5′ Phosphate, 2′-O-methyl, 2′-O-ethyl, 2′-fluoro, ribothymidine, C-5 propynyl-dC (pdC) and C-5 propynyl-dU (pdU); C-5 propynyl-C (pC) and C-5 propynyl-U (pU); 5-methyl C, 5-methyl U, 5-methyl dC, 5-methyl dU methoxy, (2,6-diaminopurine), 5′-Dimethoxytrityl-N4-ethyl-2′-deoxyCytidine and MGB (minor groove binder). It should be appreciated that more than one chemical modification can be combined within the same molecule.

Molecules associated with the invention are optimized for increased potency and/or reduced toxicity. For example, nucleotide length of the guide and/or passenger strand, and/or the number of phosphorothioate modifications in the guide and/or passenger strand, can in some aspects influence potency of the RNA molecule, while replacing 2′-fluoro (2′F) modifications with 2′-O-methyl (2′OMe) modifications can in some aspects influence toxicity of the molecule. Specifically, reduction in 2′F content of a molecule is predicted to reduce toxicity of the molecule. The Examples section presents molecules in which 2′F modifications have been eliminated, offering an advantage over previously described RNAi compounds due to a predicted reduction in toxicity. Furthermore, the number of phosphorothioate modifications in an RNA molecule can influence the uptake of the molecule into a cell, for example the efficiency of passive uptake of the molecule into a cell. Preferred embodiments of molecules described herein have no 2′F modification and yet are characterized by equal efficacy in cellular uptake and tissue penetration. Such molecules represent a significant improvement over prior art, such as molecules described by Accell and Wolfrum, which are heavily modified with extensive use of 2′F.

In some embodiments, a guide strand is approximately 18-19 nucleotides in length and has approximately 2-14 phosphate modifications. For example, a guide strand can contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more than 14 nucleotides that are phosphate-modified. The guide strand may contain one or more modifications that confer increased stability without interfering with RISC entry. The phosphate modified nucleotides, such as phosphorothioate modified nucleotides, can be at the 3′ end, 5′ end or spread throughout the guide strand. In some embodiments, the 3′ terminal 10 nucleotides of the guide strand contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 phosphorothioate modified nucleotides. The guide strand can also contain 2′F and/or 2′OMe modifications, which can be located throughout the molecule. In some embodiments, the nucleotide in position one of the guide strand (the nucleotide in the most 5′ position of the guide strand) is 2′OMe modified and/or phosphorylated. C and U nucleotides within the guide strand can be 2′F modified. For example, C and U nucleotides in positions 2-10 of a 19 nt guide strand (or corresponding positions in a guide strand of a different length) can be 2′F modified. C and U nucleotides within the guide strand can also be 2′OMe modified. For example, C and U nucleotides in positions 11-18 of a 19 nt guide strand (or corresponding positions in a guide strand of a different length) can be 2′OMe modified. In some embodiments, the nucleotide at the most 3′ end of the guide strand is unmodified. In certain embodiments, the majority of Cs and Us within the guide strand are 2′F modified and the 5′ end of the guide strand is phosphorylated. In other embodiments, position 1 and the Cs or Us in positions 11-18 are 2′OMe modified and the 5′ end of the guide strand is phosphorylated. In other embodiments, position 1 and the Cs or Us in positions 11-18 are 2′OMe modified, the 5′ end of the guide strand is phosphorylated, and the Cs or Us in position 2-10 are 2′F modified.

In some aspects, an optimal passenger strand is approximately 11-14 nucleotides in length. The passenger strand may contain modifications that confer increased stability. One or more nucleotides in the passenger strand can be 2′OMe modified. In some embodiments, one or more of the C and/or U nucleotides in the passenger strand is 2′OMe modified, or all of the C and U nucleotides in the passenger strand are 2′OMe modified. In certain embodiments, all of the nucleotides in the passenger strand are 2′OMe modified. One or more of the nucleotides on the passenger strand can also be phosphate-modified such as phosphorothioate modified. The passenger strand can also contain 2′ ribo, 2′F and 2 deoxy modifications or any combination of the above. As demonstrated in the Examples, chemical modification patterns on both the guide and passenger strand are well tolerated and a combination of chemical modifications is shown herein to lead to increased efficacy and self-delivery of RNA molecules.

Aspects of the invention relate to RNAi constructs that have extended single-stranded regions relative to double stranded regions, as compared to molecules that have been used previously for RNAi. The single stranded region of the molecules may be modified to promote cellular uptake or gene silencing. In some embodiments, phosphorothioate modification of the single stranded region influences cellular uptake and/or gene silencing. The region of the guide strand that is phosphorothioate modified can include nucleotides within both the single stranded and double stranded regions of the molecule. In some embodiments, the single stranded region includes 2-12 phosphorothioate modifications. For example, the single stranded region can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 phosphorothioate modifications. In some instances, the single stranded region contains 6-8 phosphorothioate modifications.

Molecules associated with the invention are also optimized for cellular uptake. In RNA molecules described herein, the guide and/or passenger strands can be attached to a conjugate. In certain embodiments the conjugate is hydrophobic. The hydrophobic conjugate can be a small molecule with a partition coefficient that is higher than 10. The conjugate can be a sterol-type molecule such as cholesterol, or a molecule with an increased length polycarbon chain attached to C17, and the presence of a conjugate can influence the ability of an RNA molecule to be taken into a cell with or without a lipid transfection reagent. The conjugate can be attached to the passenger or guide strand through a hydrophobic linker. In some embodiments, a hydrophobic linker is 5-12C in length, and/or is hydroxypyrrolidine-based. In some embodiments, a hydrophobic conjugate is attached to the passenger strand and the CU residues of either the passenger and/or guide strand are modified. In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the CU residues on the passenger strand and/or the guide strand are modified. In some aspects, molecules associated with the invention are self-delivering (sd). As used herein, “self-delivery” refers to the ability of a molecule to be delivered into a cell without the need for an additional delivery vehicle such as a transfection reagent.

Aspects of the invention relate to selecting molecules for use in RNAi. Molecules that have a double stranded region of 8-15 nucleotides can be selected for use in RNAi. In some embodiments, molecules are selected based on their thermodynamic stability (ΔG). In some embodiments, molecules will be selected that have a (ΔG) of less than −13 kkal/mol. For example, the (ΔG) value may be −13, −14, −15, −16, −17, −18, −19, −21, −22 or less than −22 kkal/mol. In other embodiments, the (ΔG) value may be higher than −13 kkal/mol. For example, the (ΔG) value may be −12, −11, −10, −9, −8, −7 or more than −7 kkal/mol. It should be appreciated that ΔG can be calculated using any method known in the art. In some embodiments ΔG is calculated using Mfold, available through to the Mfold internet site (http://mfold.bioinfo.rpi.edu/cgi-bin/rna-form1.cgi). Methods for calculating ΔG are described in, and are incorporated by reference from, the following references: Zuker, M. (2003) Nucleic Acids Res., 31(13):3406-15; Mathews, D. H., Sabina, J., Zuker, M. and Turner, D. H. (1999) J. Mol. Biol. 288:911-940; Mathews, D. H., Disney, M. D., Childs, J. L., Schroeder, S. J., Zuker, M., and Turner, D. H. (2004) Proc. Natl. Acad. Sci. 101:7287-7292; Duan, S., Mathews, D. H., and Turner, D. H. (2006) Biochemistry 45:9819-9832; Wuchty, S., Fontana, W., Hofacker, I. L., and Schuster, P. (1999) Biopolymers 49:145-165.

In certain embodiments, the polynucleotide contains 5′- and/or 3′-end overhangs. The number and/or sequence of nucleotides overhang on one end of the polynucleotide may be the same or different from the other end of the polynucleotide. In certain embodiments, one or more of the overhang nucleotides may contain chemical modification(s), such as phosphorothioate or 2′-OMe modification.

In certain embodiments, the polynucleotide is unmodified. In other embodiments, at least one nucleotide is modified. In further embodiments, the modification includes a 2′-H or 2′-modified ribose sugar at the 2nd nucleotide from the 5′-end of the guide sequence. The “2nd nucleotide” is defined as the second nucleotide from the 5′-end of the polynucleotide.

As used herein, “2′-modified ribose sugar” includes those ribose sugars that do not have a 2′-OH group. “2′-modified ribose sugar” does not include 2′-deoxyribose (found in unmodified canonical DNA nucleotides). For example, the 2′-modified ribose sugar may be 2′-O-alkyl nucleotides, 2′-deoxy-2′-fluoro nucleotides, 2′-deoxy nucleotides, or combination thereof.

In certain embodiments, the 2′-modified nucleotides are pyrimidine nucleotides (e.g., C/U). Examples of 2′-O-alkyl nucleotides include 2′-O-methyl nucleotides, or 2′-O-allyl nucleotides.

In certain embodiments, the sd-rxRNA polynucleotide of the invention with the above-referenced 5′-end modification exhibits significantly (e.g., at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more) less “off-target” gene silencing when compared to similar constructs without the specified 5′-end modification, thus greatly improving the overall specificity of the RNAi reagent or therapeutics.

As used herein, “off-target” gene silencing refers to unintended gene silencing due to, for example, spurious sequence homology between the antisense (guide) sequence and the unintended target mRNA sequence.

According to this aspect of the invention, certain guide strand modifications further increase nuclease stability, and/or lower interferon induction, without significantly decreasing RNAi activity (or no decrease in RNAi activity at all).

In some embodiments, wherein the RNAi construct involves a hairpin, the 5′-stem sequence may comprise a 2′-modified ribose sugar, such as 2′-O-methyl modified nucleotide, at the 2^(nd) nucleotide on the 5′-end of the polynucleotide and, in some embodiments, no other modified nucleotides. The hairpin structure having such modification may have enhanced target specificity or reduced off-target silencing compared to a similar construct without the 2′-O-methyl modification at said position.

Certain combinations of specific 5′-stem sequence and 3′-stem sequence modifications may result in further unexpected advantages, as partly manifested by enhanced ability to inhibit target gene expression, enhanced serum stability, and/or increased target specificity, etc.

In certain embodiments, the guide strand comprises a 2′-O-methyl modified nucleotide at the 2^(nd) nucleotide on the 5′-end of the guide strand and no other modified nucleotides.

In other aspects, the sd-rxRNA structures of the present invention mediates sequence-dependent gene silencing by a microRNA mechanism. As used herein, the term “microRNA” (“miRNA”), also referred to in the art as “small temporal RNAs” (“stRNAs”), refers to a small (10-50 nucleotide) RNA which are genetically encoded (e.g., by viral, mammalian, or plant genomes) and are capable of directing or mediating RNA silencing. An “miRNA disorder” shall refer to a disease or disorder characterized by an aberrant expression or activity of an miRNA.

microRNAs are involved in down-regulating target genes in critical pathways, such as development and cancer, in mice, worms and mammals. Gene silencing through a microRNA mechanism is achieved by specific yet imperfect base-pairing of the miRNA and its target messenger RNA (mRNA). Various mechanisms may be used in microRNA-mediated down-regulation of target mRNA expression.

miRNAs are noncoding RNAs of approximately 22 nucleotides which can regulate gene expression at the post transcriptional or translational level during plant and animal development. One common feature of miRNAs is that they are all excised from an approximately 70 nucleotide precursor RNA stem-loop termed pre-miRNA, probably by Dicer, an RNase III-type enzyme, or a homolog thereof. Naturally-occurring miRNAs are expressed by endogenous genes in vivo and are processed from a hairpin or stem-loop precursor (pre-miRNA or pri-miRNAs) by Dicer or other RNAses. miRNAs can exist transiently in vivo as a double-stranded duplex but only one strand is taken up by the RISC complex to direct gene silencing.

In some embodiments a version of sd-rxRNA compounds, which are effective in cellular uptake and inhibiting of miRNA activity are described. Essentially the compounds are similar to RISC entering version but large strand chemical modification patterns are optimized in the way to block cleavage and act as an effective inhibitor of the RISC action. For example, the compound might be completely or mostly Omethyl modified with the PS content described previously. For these types of compounds the 5′ phosphorylation is not necessary. The presence of double stranded region is preferred as it is promotes cellular uptake and efficient RISC loading.

Another pathway that uses small RNAs as sequence-specific regulators is the RNA interference (RNAi) pathway, which is an evolutionarily conserved response to the presence of double-stranded RNA (dsRNA) in the cell. The dsRNAs are cleaved into ˜20-base pair (bp) duplexes of small-interfering RNAs (siRNAs) by Dicer. These small RNAs get assembled into multiprotein effector complexes called RNA-induced silencing complexes (RISCs). The siRNAs then guide the cleavage of target mRNAs with perfect complementarity.

Some aspects of biogenesis, protein complexes, and function are shared between the siRNA pathway and the miRNA pathway. The subject single-stranded polynucleotides may mimic the dsRNA in the siRNA mechanism, or the microRNA in the miRNA mechanism.

In certain embodiments, the modified RNAi constructs may have improved stability in serum and/or cerebral spinal fluid compared to an unmodified RNAi constructs having the same sequence.

In certain embodiments, the structure of the RNAi construct does not induce interferon response in primary cells, such as mammalian primary cells, including primary cells from human, mouse and other rodents, and other non-human mammals. In certain embodiments, the RNAi construct may also be used to inhibit expression of a target gene in an invertebrate organism.

To further increase the stability of the subject constructs in vivo, the 3′-end of the hairpin structure may be blocked by protective group(s). For example, protective groups such as inverted nucleotides, inverted abasic moieties, or amino-end modified nucleotides may be used. Inverted nucleotides may comprise an inverted deoxynucleotide. Inverted abasic moieties may comprise an inverted deoxyabasic moiety, such as a 3′,3′-linked or 5′,5′-linked deoxyabasic moiety.

The RNAi constructs of the invention are capable of inhibiting the synthesis of any target protein encoded by target gene(s). The invention includes methods to inhibit expression of a target gene either in a cell in vitro, or in vivo. As such, the RNAi constructs of the invention are useful for treating a patient with a disease characterized by the overexpression of a target gene.

The target gene can be endogenous or exogenous (e.g., introduced into a cell by a virus or using recombinant DNA technology) to a cell. Such methods may include introduction of RNA into a cell in an amount sufficient to inhibit expression of the target gene. By way of example, such an RNA molecule may have a guide strand that is complementary to the nucleotide sequence of the target gene, such that the composition inhibits expression of the target gene.

The invention also relates to vectors expressing the subject hairpin constructs, and cells comprising such vectors or the subject hairpin constructs. The cell may be a mammalian cell in vivo or in culture, such as a human cell.

The invention further relates to compositions comprising the subject RNAi constructs, and a pharmaceutically acceptable carrier or diluent.

Another aspect of the invention provides a method for inhibiting the expression of a target gene in a mammalian cell, comprising contacting the mammalian cell with any of the subject RNAi constructs.

The method may be carried out in vitro, ex vivo, or in vivo, in, for example, mammalian cells in culture, such as a human cell in culture.

The target cells (e.g., mammalian cell) may be contacted in the presence of a delivery reagent, such as a lipid (e.g., a cationic lipid) or a liposome.

Another aspect of the invention provides a method for inhibiting the expression of a target gene in a mammalian cell, comprising contacting the mammalian cell with a vector expressing the subject RNAi constructs.

In one aspect of the invention, a longer duplex polynucleotide is provided, including a first polynucleotide that ranges in size from about 16 to about 30 nucleotides; a second polynucleotide that ranges in size from about 26 to about 46 nucleotides, wherein the first polynucleotide (the antisense strand) is complementary to both the second polynucleotide (the sense strand) and a target gene, and wherein both polynucleotides form a duplex and wherein the first polynucleotide contains a single stranded region longer than 6 bases in length and is modified with alternative chemical modification pattern, and/or includes a conjugate moiety that facilitates cellular delivery. In this embodiment, between about 40% to about 90% of the nucleotides of the passenger strand between about 40% to about 90% of the nucleotides of the guide strand, and between about 40% to about 90% of the nucleotides of the single stranded region of the first polynucleotide are chemically modified nucleotides.

In an embodiment, the chemically modified nucleotide in the polynucleotide duplex may be any chemically modified nucleotide known in the art, such as those discussed in detail above. In a particular embodiment, the chemically modified nucleotide is selected from the group consisting of 2′ F modified nucleotides, 2′-O-methyl modified and 2′deoxy nucleotides. In another particular embodiment, the chemically modified nucleotides results from “hydrophobic modifications” of the nucleotide base. In another particular embodiment, the chemically modified nucleotides are phosphorothioates. In an additional particular embodiment, chemically modified nucleotides are combination of phosphorothioates, 2′-O-methyl, 2′deoxy, hydrophobic modifications and phosphorothioates. As these groups of modifications refer to modification of the ribose ring, back bone and nucleotide, it is feasible that some modified nucleotides will carry a combination of all three modification types.

In another embodiment, the chemical modification is not the same across the various regions of the duplex. In a particular embodiment, the first polynucleotide (the passenger strand), has a large number of diverse chemical modifications in various positions. For this polynucleotide up to 90% of nucleotides might be chemically modified and/or have mismatches introduced. In another embodiment, chemical modifications of the first or second polynucleotide include, but not limited to, 5′ position modification of Uridine and Cytosine (4-pyridyl, 2-pyridyl, indolyl, phenyl (C₆H₅OH); tryptophanyl (C8H6N)CH2CH(NH2)CO), isobutyl, butyl, aminobenzyl; phenyl; naphthyl, etc), where the chemical modification might alter base pairing capabilities of a nucleotide. For the guide strand an important feature of this aspect of the invention is the position of the chemical modification relative to the 5′ end of the antisense and sequence. For example, chemical phosphorylation of the 5′ end of the guide strand is usually beneficial for efficacy. O-methyl modifications in the seed region of the sense strand (position 2-7 relative to the 5′ end) are not generally well tolerated, whereas 2′F and deoxy are well tolerated. The mid part of the guide strand and the 3′ end of the guide strand are more permissive in a type of chemical modifications applied. Deoxy modifications are not tolerated at the 3′ end of the guide strand.

A unique feature of this aspect of the invention involves the use of hydrophobic modification on the bases. In one embodiment, the hydrophobic modifications are preferably positioned near the 5′ end of the guide strand, in other embodiments, they localized in the middle of the guides strand, in other embodiment they localized at the 3′ end of the guide strand and yet in another embodiment they are distributed thought the whole length of the polynucleotide. The same type of patterns is applicable to the passenger strand of the duplex.

The other part of the molecule is a single stranded region. The single stranded region is expected to range from 6 to 40 nucleotides. In one embodiment, the single stranded region of the first polynucleotide contains modifications selected from the group consisting of between 40% and 90% hydrophobic base modifications, between 40%-90% phosphorothioates, between 40%-90% modification of the ribose moiety, and any combination of the preceding.

Efficiency of guide strand (first polynucleotide) loading into the RISC complex might be altered for heavily modified polynucleotides, so in one embodiment, the duplex polynucleotide includes a mismatch between nucleotide 9, 11, 12, 13, or 14 on the guide strand (first polynucleotide) and the opposite nucleotide on the sense strand (second polynucleotide) to promote efficient guide strand loading.

More detailed aspects of the invention are described in the sections below.

Duplex Characteristics

Double-stranded oligonucleotides of the invention may be formed by two separate complementary nucleic acid strands. Duplex formation can occur either inside or outside the cell containing the target gene.

As used herein, the term “duplex” includes the region of the double-stranded nucleic acid molecule(s) that is (are) hydrogen bonded to a complementary sequence. Double-stranded oligonucleotides of the invention may comprise a nucleotide sequence that is sense to a target gene and a complementary sequence that is antisense to the target gene. The sense and antisense nucleotide sequences correspond to the target gene sequence, e.g., are identical or are sufficiently identical to effect target gene inhibition (e.g., are about at least about 98% identical, 96% identical, 94%, 90% identical, 85% identical, or 80% identical) to the target gene sequence.

In certain embodiments, the double-stranded oligonucleotide of the invention is double-stranded over its entire length, i.e., with no overhanging single-stranded sequence at either end of the molecule, i.e., is blunt-ended. In other embodiments, the individual nucleic acid molecules can be of different lengths. In other words, a double-stranded oligonucleotide of the invention is not double-stranded over its entire length. For instance, when two separate nucleic acid molecules are used, one of the molecules, e.g., the first molecule comprising an antisense sequence, can be longer than the second molecule hybridizing thereto (leaving a portion of the molecule single-stranded). Likewise, when a single nucleic acid molecule is used a portion of the molecule at either end can remain single-stranded.

In one embodiment, a double-stranded oligonucleotide of the invention contains mismatches and/or loops or bulges, but is double-stranded over at least about 70% of the length of the oligonucleotide. In another embodiment, a double-stranded oligonucleotide of the invention is double-stranded over at least about 80% of the length of the oligonucleotide. In another embodiment, a double-stranded oligonucleotide of the invention is double-stranded over at least about 90%-95% of the length of the oligonucleotide. In another embodiment, a double-stranded oligonucleotide of the invention is double-stranded over at least about 96%-98% of the length of the oligonucleotide. In certain embodiments, the double-stranded oligonucleotide of the invention contains at least or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mismatches.

Modifications

The nucleotides of the invention may be modified at various locations, including the sugar moiety, the phosphodiester linkage, and/or the base.

In some embodiments, the base moiety of a nucleoside may be modified. For example, a pyrimidine base may be modified at the 2, 3, 4, 5, and/or 6 position of the pyrimidine ring. In some embodiments, the exocyclic amine of cytosine may be modified. A purine base may also be modified. For example, a purine base may be modified at the 1, 2, 3, 6, 7, or 8 position. In some embodiments, the exocyclic amine of adenine may be modified. In some cases, a nitrogen atom in a ring of a base moiety may be substituted with another atom, such as carbon. A modification to a base moiety may be any suitable modification. Examples of modifications are known to those of ordinary skill in the art. In some embodiments, the base modifications include alkylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocycles.

In some embodiments, a pyrimidine may be modified at the 5 position. For example, the 5 position of a pyrimidine may be modified with an alkyl group, an alkynyl group, an alkenyl group, an acyl group, or substituted derivatives thereof. In other examples, the 5 position of a pyrimidine may be modified with a hydroxyl group or an alkoxyl group or substituted derivative thereof. Also, the N⁴ position of a pyrimidine may be alkylated. In still further examples, the pyrimidine 5-6 bond may be saturated, a nitrogen atom within the pyrimidine ring may be substituted with a carbon atom, and/or the O² and O⁴ atoms may be substituted with sulfur atoms. It should be understood that other modifications are possible as well.

In other examples, the N⁷ position and/or N² and/or N³ position of a purine may be modified with an alkyl group or substituted derivative thereof. In further examples, a third ring may be fused to the purine bicyclic ring system and/or a nitrogen atom within the purine ring system may be substituted with a carbon atom. It should be understood that other modifications are possible as well.

Non-limiting examples of pyrimidines modified at the 5 position are disclosed in U.S. Pat. Nos. 5,591,843, 7,205,297, 6,432,963, and 6,020,483; non-limiting examples of pyrimidines modified at the N⁴ position are disclosed in U.S. Pat. No. 5,580,731; non-limiting examples of purines modified at the 8 position are disclosed in U.S. Pat. Nos. 6,355,787 and 5,580,972; non-limiting examples of purines modified at the N position are disclosed in U.S. Pat. Nos. 4,853,386, 5,789,416, and 7,041,824; and non-limiting examples of purines modified at the 2 position are disclosed in U.S. Pat. Nos. 4,201,860 and 5,587,469, all of which are incorporated herein by reference.

Non-limiting examples of modified bases include N⁴,N⁴-ethanocytosine, 7-deazaxanthosine, 7-deazaguanosine, 8-oxo-N⁶-methyladenine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyl uracil, dihydrouracil, inosine, N⁶-isopentenyl-adenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N⁶-methyladenine, 7-methylguanine, 5-methylaminomethyl uracil, 5-methoxy aminomethyl-2-thiouracil, 5-methoxyuracil, 2-methylthio-N⁶-isopentenyladenine, pseudouracil, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, 2-thiocytosine, and 2,6-diaminopurine. In some embodiments, the base moiety may be a heterocyclic base other than a purine or pyrimidine. The heterocyclic base may be optionally modified and/or substituted.

Sugar moieties include natural, unmodified sugars, e.g., monosaccharide (such as pentose, e.g., ribose, deoxyribose), modified sugars and sugar analogs. In general, possible modifications of nucleomonomers, particularly of a sugar moiety, include, for example, replacement of one or more of the hydroxyl groups with a halogen, a heteroatom, an aliphatic group, or the functionalization of the hydroxyl group as an ether, an amine, a thiol, or the like.

One particularly useful group of modified nucleomonomers are 2′-O-methyl nucleotides. Such 2′-O-methyl nucleotides may be referred to as “methylated,” and the corresponding nucleotides may be made from unmethylated nucleotides followed by alkylation or directly from methylated nucleotide reagents. Modified nucleomonomers may be used in combination with unmodified nucleomonomers. For example, an oligonucleotide of the invention may contain both methylated and unmethylated nucleomonomers.

Some exemplary modified nucleomonomers include sugar- or backbone-modified ribonucleotides. Modified ribonucleotides may contain a non-naturally occurring base (instead of a naturally occurring base), such as uridines or cytidines modified at the 5′-position, e.g., 5′-(2-amino)propyl uridine and 5′-bromo uridine; adenosines and guanosines modified at the 8-position, e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; and N-alkylated nucleotides, e.g., N6-methyl adenosine. Also, sugar-modified ribonucleotides may have the 2′-OH group replaced by a H, alxoxy (or OR), R or alkyl, halogen, SH, SR, amino (such as NH₂, NHR, NR₂), or CN group, wherein R is lower alkyl, alkenyl, or alkynyl.

Modified ribonucleotides may also have the phosphodiester group connecting to adjacent ribonucleotides replaced by a modified group, e.g., of phosphorothioate group. More generally, the various nucleotide modifications may be combined.

Although the antisense (guide) strand may be substantially identical to at least a portion of the target gene (or genes), at least with respect to the base pairing properties, the sequence need not be perfectly identical to be useful, e.g., to inhibit expression of a target gene's phenotype. Generally, higher homology can be used to compensate for the use of a shorter antisense gene. In some cases, the antisense strand generally will be substantially identical (although in antisense orientation) to the target gene.

The use of 2′-O-methyl modified RNA may also be beneficial in circumstances in which it is desirable to minimize cellular stress responses. RNA having 2′-O-methyl nucleomonomers may not be recognized by cellular machinery that is thought to recognize unmodified RNA. The use of 2′-O-methylated or partially 2′-O-methylated RNA may avoid the interferon response to double-stranded nucleic acids, while maintaining target RNA inhibition. This may be useful, for example, for avoiding the interferon or other cellular stress responses, both in short RNAi (e.g., siRNA) sequences that induce the interferon response, and in longer RNAi sequences that may induce the interferon response.

Overall, modified sugars may include D-ribose, 2′-O-alkyl (including 2′-O-methyl and 2′-O-ethyl), i.e., 2′-alkoxy, 2′-amino, 2′-S-alkyl, 2′-halo (including 2′-fluoro), 2′-methoxyethoxy, 2′-allyloxy (—OCH₂CH═CH₂), 2′-propargyl, 2′-propyl, ethynyl, ethenyl, propenyl, and cyano and the like. In one embodiment, the sugar moiety can be a hexose and incorporated into an oligonucleotide as described (Augustyns, K., et al., Nucl. Acids. Res. 18:4711 (1992)). Exemplary nucleomonomers can be found, e.g., in U.S. Pat. No. 5,849,902, incorporated by reference herein.

Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75^(th) Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito: 1999, the entire contents of which are incorporated herein by reference.

Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention.

Isomeric mixtures containing any of a variety of isomer ratios may be utilized in accordance with the present invention. For example, where only two isomers are combined, mixtures containing 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0 isomer ratios are all contemplated by the present invention. Those of ordinary skill in the art will readily appreciate that analogous ratios are contemplated for more complex isomer mixtures.

If, for instance, a particular enantiomer of a compound of the present invention is desired, it may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts are formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers.

In certain embodiments, oligonucleotides of the invention comprise 3′ and 5′ termini (except for circular oligonucleotides). In one embodiment, the 3′ and 5′ termini of an oligonucleotide can be substantially protected from nucleases e.g., by modifying the 3′ or 5′ linkages (e.g., U.S. Pat. No. 5,849,902 and WO 98/13526). For example, oligonucleotides can be made resistant by the inclusion of a “blocking group.” The term “blocking group” as used herein refers to substituents (e.g., other than OH groups) that can be attached to oligonucleotides or nucleomonomers, either as protecting groups or coupling groups for synthesis (e.g., FITC, propyl (CH₂—CH₂—CH₃), glycol (—O—CH₂—CH₂—O—) phosphate (PO₃ ²⁻), hydrogen phosphonate, or phosphoramidite). “Blocking groups” also include “end blocking groups” or “exonuclease blocking groups” which protect the 5′ and 3′ termini of the oligonucleotide, including modified nucleotides and non-nucleotide exonuclease resistant structures.

Exemplary end-blocking groups include cap structures (e.g., a 7-methylguanosine cap), inverted nucleomonomers, e.g., with 3′-3′ or 5′-5′ end inversions (see, e.g., Ortiagao et al. 1992. Antisense Res. Dev. 2:129), methylphosphonate, phosphoramidite, non-nucleotide groups (e.g., non-nucleotide linkers, amino linkers, conjugates) and the like. The 3′ terminal nucleomonomer can comprise a modified sugar moiety. The 3′ terminal nucleomonomer comprises a 3′-O that can optionally be substituted by a blocking group that prevents 3′-exonuclease degradation of the oligonucleotide. For example, the 3′-hydroxyl can be esterified to a nucleotide through a 3′→3′ internucleotide linkage. For example, the alkyloxy radical can be methoxy, ethoxy, or isopropoxy, and preferably, ethoxy. Optionally, the 3′→3′linked nucleotide at the 3′ terminus can be linked by a substitute linkage. To reduce nuclease degradation, the 5′ most 3′→5′ linkage can be a modified linkage, e.g., a phosphorothioate or a P-alkyloxyphosphotriester linkage. Preferably, the two 5′ most 3′→5′ linkages are modified linkages. Optionally, the 5′ terminal hydroxy moiety can be esterified with a phosphorus containing moiety, e.g., phosphate, phosphorothioate, or P-ethoxyphosphate.

One of ordinary skill in the art will appreciate that the synthetic methods, as described herein, utilize a variety of protecting groups. By the term “protecting group,” as used herein, it is meant that a particular functional moiety, e.g., O, S, or N, is temporarily blocked so that a reaction can be carried out selectively at another reactive site in a multifunctional compound. In certain embodiments, a protecting group reacts selectively in good yield to give a protected substrate that is stable to the projected reactions; the protecting group should be selectively removable in good yield by readily available, preferably non-toxic reagents that do not attack the other functional groups; the protecting group forms an easily separable derivative (more preferably without the generation of new stereogenic centers); and the protecting group has a minimum of additional functionality to avoid further sites of reaction. As detailed herein, oxygen, sulfur, nitrogen, and carbon protecting groups may be utilized. Hydroxyl protecting groups include methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido, diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, a-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl, 4.4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4′,4″-tris(levulinoyloxyphenyl)methyl, 4,4′,4″-tris(benzoyloxyphenyl)methyl, 3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl, 1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate, adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec), 2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate, o-(methoxycarbonyl)benzoate, a-naphthoate, nitrate, alkyl N,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts). For protecting 1,2- or 1,3-diols, the protecting groups include methylene acetal, ethylidene acetal, 1-t-butylethylidene ketal, 1-phenylethylidene ketal. (4-methoxyphenyl)ethylidene acetal. 2,2,2-trichloroethylidene acetal, acetonide, cyclopentylidene ketal, cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal, p-methoxybenzylidene acetal, 2,4-dimethoxybenzylidene ketal, 3,4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal, methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethylene ortho ester, 1-methoxyethylidene ortho ester, 1-ethoxyethylidine ortho ester, 1,2-dimethoxyethylidene ortho ester, a-methoxybenzylidene ortho ester, 1-(N,N-dimethylamino)ethylidene derivative, a-(N,N′-dimethylamino)benzylidene derivative, 2-oxacyclopentylidene ortho ester, di-t-butylsilylene group (DTBS), 1,3-(1,1,3,3-tetraisopropyldisiloxanylidene) derivative (TIPDS), tetra-t-butoxydisiloxane-1,3-diylidene derivative (TBDS), cyclic carbonates, cyclic boronates, ethyl boronate, and phenyl boronate. Amino-protecting groups include methyl carbamate, ethyl carbamante, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and 4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate. N-hydroxypiperidinyl carbamate, alkyldithio carbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methyl carbamate, phenothiazinyl-(10)-carbonyl derivative, N′-p-toluenesulfonylaminocarbonyl derivative, N′-phenylaminothiocarbonyl derivative, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxycarbonylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzyl carbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, 2,4,6-trimethylbenzyl carbamate, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide, p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (N′-dithiobenzyloxycarbonylamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine derivative, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF). N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), N-2-picolylamino N′-oxide. N-1,1-dimethylthiomethyleneamine, N-benzylideneamine, N-p-methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N—(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine, N-p-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine, N-borane derivative, N-diphenylborinic acid derivative, N-[phenyl(pentacarbonylchromium- or tungsten)carbonyl]amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (Npys), p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), 3-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide. Exemplary protecting groups are detailed herein. However, it will be appreciated that the present invention is not intended to be limited to these protecting groups; rather, a variety of additional equivalent protecting groups can be readily identified using the above criteria and utilized in the method of the present invention. Additionally, a variety of protecting groups are described in Protective Groups in Organic Synthesis, Third Ed. Greene, T. W. and Wuts, P. G., Eds., John Wiley & Sons, New York: 1999, the entire contents of which are hereby incorporated by reference.

It will be appreciated that the compounds, as described herein, may be substituted with any number of substituents or functional moieties. In general, the term “substituted” whether proceeded by the term “optionally” or not, and substituents contained in formulas of this invention, refer to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent. When more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms. Furthermore, this invention is not intended to be limited in any manner by the permissible substituents of organic compounds. Combinations of substituents and variables envisioned by this invention are preferably those that result in the formation of stable compounds useful in the treatment, for example, of infectious diseases or proliferative disorders. The term “stable”, as used herein, preferably refers to compounds which possess stability sufficient to allow manufacture and which maintain the integrity of the compound for a sufficient period of time to be detected and preferably for a sufficient period of time to be useful for the purposes detailed herein.

The term “aliphatic,” as used herein, includes both saturated and unsaturated, straight chain (i.e., unbranched), branched, acyclic, cyclic, or polycyclic aliphatic hydrocarbons, which are optionally substituted with one or more functional groups. As will be appreciated by one of ordinary skill in the art, “aliphatic” is intended herein to include, but is not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties. Thus, as used herein, the term “alkyl” includes straight, branched and cyclic alkyl groups. An analogous convention applies to other generic terms such as “alkenyl,” “alkynyl,” and the like. Furthermore, as used herein, the terms “alkyl,” “alkenyl,” “alkynyl,” and the like encompass both substituted and unsubstituted groups. In certain embodiments, as used herein. “lower alkyl” is used to indicate those alkyl groups (cyclic, acyclic, substituted, unsubstituted, branched, or unbranched) having 1-6 carbon atoms.

In certain embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-20 aliphatic carbon atoms. In certain other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-10 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-8 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-6 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-4 carbon atoms. Illustrative aliphatic groups thus include, but are not limited to, for example, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, —CH₂— cyclopropyl, vinyl, allyl, n-butyl, sec-butyl, isobutyl, tert-butyl, cyclobutyl, —CH₂— cyclobutyl, n-pentyl, sec-pentyl, isopentyl, tert-pentyl, cyclopentyl, —CH₂-cyclopentyl, n-hexyl, sec-hexyl, cyclohexyl, —CH₂-cyclohexyl moieties and the like, which again, may bear one or more substituents. Alkenyl groups include, but are not limited to, for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like. Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like.

Some examples of substituents of the above-described aliphatic (and other) moieties of compounds of the invention include, but are not limited to aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; —F: —Cl; —Br; —I; —OH; —NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂; —CH₂SO₂CH₃; —C(O)R_(x); —CO₂(R_(x)); —CON(R_(x))₂; —OC(O)R_(x); —OCO₂R_(x): —OCON(R_(x))₂; —N(R_(x))₂; —S(O)₂R_(x); —NR_(x)(CO)R_(x) wherein each occurrence of R_(x) independently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, wherein any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and wherein any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Additional examples of generally applicable substituents are illustrated by the specific embodiments described herein.

The term “heteroaliphatic,” as used herein, refers to aliphatic moieties that contain one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms, e.g., in place of carbon atoms. Heteroaliphatic moieties may be branched, unbranched, cyclic or acyclic and include saturated and unsaturated heterocycles such as morpholino, pyrrolidinyl, etc. In certain embodiments, heteroaliphatic moieties are substituted by independent replacement of one or more of the hydrogen atoms thereon with one or more moieties including, but not limited to aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; —F; —Cl; —Br; —I; —OH; —NO₂: —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂; —CH₂SO₂CH₃; —C(O)R_(x); —CO₂(R_(x)); —CON(R_(x))₂; —OC(O)R_(x); —OCO₂R_(x); —OCON(R_(x))₂; —N(R_(x))₂; —S(O)₂R_(x); —NR_(x)(CO)R_(x), wherein each occurrence of R_(x) independently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, wherein any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and wherein any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Additional examples of generally applicable substitutents are illustrated by the specific embodiments described herein.

The terms “halo” and “halogen” as used herein refer to an atom selected from fluorine, chlorine, bromine, and iodine.

The term “alkyl” includes saturated aliphatic groups, including straight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups (isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups (cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In certain embodiments, a straight chain or branched chain alkyl has 6 or fewer carbon atoms in its backbone (e.g., C₁-C₆ for straight chain, C₃-C₆ for branched chain), and more preferably 4 or fewer. Likewise, preferred cycloalkyls have from 3-8 carbon atoms in their ring structure, and more preferably have 5 or 6 carbons in the ring structure. The term C₁-C₆ includes alkyl groups containing 1 to 6 carbon atoms.

Moreover, unless otherwise specified, the term alkyl includes both “unsubstituted alkyls” and “substituted alkyls,” the latter of which refers to alkyl moieties having independently selected substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Cycloalkyls can be further substituted, e.g., with the substituents described above. An “alkylaryl” or an “arylalkyl” moiety is an alkyl substituted with an aryl (e.g., phenylmethyl (benzyl)). The term “alkyl” also includes the side chains of natural and unnatural amino acids. The term “n-alkyl” means a straight chain (i.e., unbranched) unsubstituted alkyl group.

The term “alkenyl” includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double bond. For example, the term “alkenyl” includes straight-chain alkenyl groups (e.g., ethylenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, etc.), branched-chain alkenyl groups, cycloalkenyl (alicyclic) groups (cyclopropenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl), alkyl or alkenyl substituted cycloalkenyl groups, and cycloalkyl or cycloalkenyl substituted alkenyl groups. In certain embodiments, a straight chain or branched chain alkenyl group has 6 or fewer carbon atoms in its backbone (e.g., C₂-C₆ for straight chain, C₃-C₆ for branched chain). Likewise, cycloalkenyl groups may have from 3-8 carbon atoms in their ring structure, and more preferably have 5 or 6 carbons in the ring structure. The term C₂-C₆ includes alkenyl groups containing 2 to 6 carbon atoms.

Moreover, unless otherwise specified, the term alkenyl includes both “unsubstituted alkenyls” and “substituted alkenyls,” the latter of which refers to alkenyl moieties having independently selected substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, alkyl groups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulthydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

The term “alkynyl” includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but which contain at least one triple bond. For example, the term “alkynyl” includes straight-chain alkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, etc.), branched-chain alkynyl groups, and cycloalkyl or cycloalkenyl substituted alkynyl groups. In certain embodiments, a straight chain or branched chain alkynyl group has 6 or fewer carbon atoms in its backbone (e.g., C₂-C₆ for straight chain, C₃-C₆ for branched chain). The term C₂-C₆ includes alkynyl groups containing 2 to 6 carbon atoms.

Moreover, unless otherwise specified, the term alkynyl includes both “unsubstituted alkynyls” and “substituted alkynyls,” the latter of which refers to alkynyl moieties having independently selected substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, alkyl groups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

Unless the number of carbons is otherwise specified, “lower alkyl” as used herein means an alkyl group, as defined above, but having from one to five carbon atoms in its backbone structure. “Lower alkenyl” and “lower alkynyl” have chain lengths of, for example, 2-5 carbon atoms.

The term “alkoxy” includes substituted and unsubstituted alkyl, alkenyl, and alkynyl groups covalently linked to an oxygen atom. Examples of alkoxy groups include methoxy, ethoxy, isopropyloxy, propoxy, butoxy, and pentoxy groups. Examples of substituted alkoxy groups include halogenated alkoxy groups. The alkoxy groups can be substituted with independently selected groups such as alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulffiydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moieties. Examples of halogen substituted alkoxy groups include, but are not limited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy, trichloromethoxy, etc.

The term “hydrophobic modifications’ include bases modified in a fashion, where (1) overall hydrophobicity of the base is significantly increases, (2) the base is still capable of forming close to regular Watson-Crick interaction. Some, of the examples of base modifications include but are not limited to 5-position uridine and cytidine modifications like phenyl.

4-pyridyl, 2-pyridyl, indolyl, and isobutyl, phenyl (C6H5OH); tryptophanyl (C8H6N)CH2CH(NH2)CO), Isobutyl, butyl, aminobenzyl; phenyl; naphthyl, For purposes of the present invention, the term “overhang” refers to terminal non-base pairing nucleotide(s) resulting from one strand or region extending beyond the terminus of the complementary strand to which the first strand or region forms a duplex. One or more polynucleotides that are capable of forming a duplex through hydrogen bonding can have overhangs. The overhand length generally doesn't exceed 5 bases in length.

The term “heteroatom” includes atoms of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur and phosphorus.

The term “hydroxy” or “hydroxyl” includes groups with an —OH or —O⁻ (with an appropriate counterion).

The term “halogen” includes fluorine, bromine, chlorine, iodine, etc. The term “perhalogenated” generally refers to a moiety wherein all hydrogens are replaced by halogen atoms.

The term “substituted” includes independently selected substituents which can be placed on the moiety and which allow the molecule to perform its intended function. Examples of substituents include alkyl, alkenyl, alkynyl, aryl, (CR′R″)₀₋₃NR′R″, (CR′R″)₀₋₃CN, NO₂, halogen, (CR′R″)₀₋₃C(halogen)₃, (CR′R″)₀₋₃CH(halogen)₂, (CR′R″)₀₋₃CH₂(halogen), (CR′R″)₀₋₃CONR′R″, (CR′R″)₀₋₃S(O)₁₋₂NR′R″, (CR′R″)₀₋₃CHO, (CR′R″)₀₋₃O(CR′R″)₀₋₃H, (CR′R″)₀₋₃S(O)₀₋₂R′, (CR′R″)₀₋₃O(CR′R″)₀₋₃H, (CR′R″)₀₋₃COR′, (CR′R″)₀₋₃CO₂R′, or (CR′R″)₀₋₃OR′ groups; wherein each R′ and R″ are each independently hydrogen, a C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅ alkynyl, or aryl group, or R′ and R″ taken together are a benzylidene group or a —CH₂)₂O(CH₂)₂— group.

The term “amine” or “amino” includes compounds or moieties in which a nitrogen atom is covalently bonded to at least one carbon or heteroatom. The term “alkyl amino” includes groups and compounds wherein the nitrogen is bound to at least one additional alkyl group. The term “dialkyl amino” includes groups wherein the nitrogen atom is bound to at least two additional alkyl groups.

The term “ether” includes compounds or moieties which contain an oxygen bonded to two different carbon atoms or heteroatoms. For example, the term includes “alkoxyalkyl,” which refers to an alkyl, alkenyl, or alkynyl group covalently bonded to an oxygen atom which is covalently bonded to another alkyl group.

The terms “polynucleotide,” “nucleotide sequence,” “nucleic acid,” “nucleic acid molecule,” “nucleic acid sequence,” and “oligonucleotide” refer to a polymer of two or more nucleotides. The polynucleotides can be DNA, RNA, or derivatives or modified versions thereof. The polynucleotide may be single-stranded or double-stranded. The polynucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, its hybridization parameters, etc. The polynucleotide may comprise a modified base moiety which is selected from the group including but not limited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, I-methylguanine, I-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine. The polynucleotide may comprise a modified sugar moiety (e.g., 2′-fluororibose, ribose, 2′-deoxyribose, 2′-O-methylcytidine, arabinose, and hexose), and/or a modified phosphate moiety (e.g., phosphorothioates and 5′-N-phosphoramidite linkages). A nucleotide sequence typically carries genetic information, including the information used by cellular machinery to make proteins and enzymes. These terms include double- or single-stranded genomic and cDNA, RNA, any synthetic and genetically manipulated polynucleotide, and both sense and antisense polynucleotides. This includes single- and double-stranded molecules, i.e., DNA-DNA. DNA-RNA, and RNA-RNA hybrids, as well as “protein nucleic acids” (PNA) formed by conjugating bases to an amino acid backbone.

The term “base” includes the known purine and pyrimidine heterocyclic bases, deazapurines, and analogs (including heterocyclic substituted analogs, e.g., aminoethyoxy phenoxazine), derivatives (e.g., 1-alkyl-, 1-alkenyl-, heteroaromatic- and 1-alkynyl derivatives) and tautomers thereof. Examples of purines include adenine, guanine, inosine, diaminopurine, and xanthine and analogs (e.g., 8-oxo-N⁶-methyladenine or 7-diazaxanthine) and derivatives thereof. Pyrimidines include, for example, thymine, uracil, and cytosine, and their analogs (e.g., 5-methylcytosine, 5-methyluracil, 5-(1-propynyl)uracil, 5-(1-propynyl)cytosine and 4,4-ethanocytosine). Other examples of suitable bases include non-purinyl and non-pyrimidinyl bases such as 2-aminopyridine and triazines.

In a preferred embodiment, the nucleomonomers of an oligonucleotide of the invention are RNA nucleotides. In another preferred embodiment, the nucleomonomers of an oligonucleotide of the invention are modified RNA nucleotides. Thus, the oligonucleotides contain modified RNA nucleotides.

The term “nucleoside” includes bases which are covalently attached to a sugar moiety, preferably ribose or deoxyribose. Examples of preferred nucleosides include ribonucleosides and deoxyribonucleosides. Nucleosides also include bases linked to amino acids or amino acid analogs which may comprise free carboxyl groups, free amino groups, or protecting groups. Suitable protecting groups are well known in the art (see P. G. M. Wuts and T. W. Greene, “Protective Groups in Organic Synthesis”, 2^(nd) Ed., Wiley-Interscience, New York, 1999).

The term “nucleotide” includes nucleosides which further comprise a phosphate group or a phosphate analog.

The nucleic acid molecules may be associated with a hydrophobic moiety for targeting and/or delivery of the molecule to a cell. In certain embodiments, the hydrophobic moiety is associated with the nucleic acid molecule through a linker. In certain embodiments, the association is through non-covalent interactions. In other embodiments, the association is through a covalent bond. Any linker known in the art may be used to associate the nucleic acid with the hydrophobic moiety. Linkers known in the art are described in published international PCT applications, WO 92/03464. WO 95/23162, WO 2008/021157, WO 2009/021157, WO 2009/134487, WO 2009/126933, U.S. Patent Application Publication 2005/0107325, U.S. Pat. Nos. 5,414,077, 5,419,966, 5,512,667, 5,646,126, and 5,652,359, which are incorporated herein by reference. The linker may be as simple as a covalent bond to a multi-atom linker. The linker may be cyclic or acyclic. The linker may be optionally substituted. In certain embodiments, the linker is capable of being cleaved from the nucleic acid. In certain embodiments, the linker is capable of being hydrolyzed under physiological conditions. In certain embodiments, the linker is capable of being cleaved by an enzyme (e.g., an esterase or phosphodiesterase). In certain embodiments, the linker comprises a spacer element to separate the nucleic acid from the hydrophobic moiety. The spacer element may include one to thirty carbon or heteroatoms. In certain embodiments, the linker and/or spacer element comprises protonatable functional groups. Such protonatable functional groups may promote the endosomal escape of the nucleic acid molecule. The protonatable functional groups may also aid in the delivery of the nucleic acid to a cell, for example, neutralizing the overall charge of the molecule. In other embodiments, the linker and/or spacer element is biologically inert (that is, it does not impart biological activity or function to the resulting nucleic acid molecule).

In certain embodiments, the nucleic acid molecule with a linker and hydrophobic moiety is of the formulae described herein. In certain embodiments, the nucleic acid molecule is of the formula:

wherein X is N or CH; A is a bond; substituted or unsubstituted, cyclic or acyclic, branched or unbranched aliphatic; or substituted or unsubstituted, cyclic or acyclic, branched or unbranched heteroaliphatic; R¹ is a hydrophobic moiety; R² is hydrogen; an oxygen-protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; and R³ is a nucleic acid. In certain embodiments, the molecule is of the formula:

In certain embodiments, the molecule is of the formula:

In certain embodiments, the molecule is of the formula:

In certain embodiments, the molecule is of the formula:

In certain embodiments, X is N. In certain embodiments, X is CH. In certain embodiments, A is a bond. In certain embodiments, A is substituted or unsubstituted, cyclic or acyclic, branched or unbranched aliphatic. In certain embodiments. A is acyclic, substituted or unsubstituted, branched or unbranched aliphatic. In certain embodiments, A is acyclic, substituted, branched or unbranched aliphatic. In certain embodiments, A is acyclic, substituted, unbranched aliphatic. In certain embodiments. A is acyclic, substituted, unbranched alkyl. In certain embodiments, A is acyclic, substituted, unbranched C₁₋₂₀ alkyl. In certain embodiments, A is acyclic, substituted, unbranched C₁₋₁₂ alkyl. In certain embodiments, A is acyclic, substituted, unbranched C₁₋₁₀ alkyl. In certain embodiments, A is acyclic, substituted, unbranched C₁₋₈ alkyl. In certain embodiments, A is acyclic, substituted, unbranched C₁₋₆ alkyl. In certain embodiments, A is substituted or unsubstituted, cyclic or acyclic, branched or unbranched heteroaliphatic. In certain embodiments, A is acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic. In certain embodiments, A is acyclic, substituted, branched or unbranched heteroaliphatic. In certain embodiments. A is acyclic, substituted, unbranched heteroaliphatic. In certain embodiments, A is of the formula:

In certain embodiments, A is of one of the formulae:

In certain embodiments, A is of one of the formulae:

In certain embodiments, A is of one of the formulae:

In certain embodiments, A is of the formula:

In certain embodiments, A is of the formula:

In certain embodiments, A is of the formula:

wherein each occurrence of R is independently the side chain of a natural or unnatural amino acid; and n is an integer between 1 and 20, inclusive. In certain embodiments, A is of the formula:

In certain embodiments, each occurrence of R is independently the side chain of a natural amino acid. In certain embodiments, n is an integer between 1 and 15, inclusive. In certain embodiments, n is an integer between 1 and 10, inclusive. In certain embodiments, n is an integer between 1 and 5, inclusive. In certain embodiments, A is of the formula:

wherein n is an integer between 1 and 20, inclusive. In certain embodiments, A is of the formula:

In certain embodiments, n is an integer between 1 and 15, inclusive. In certain embodiments, n is an integer between 1 and 10, inclusive. In certain embodiments, n is an integer between 1 and 5, inclusive. In certain embodiments. A is of the formula:

wherein n is an integer between 1 and 20, inclusive. In certain embodiments, A is of the formula:

In certain embodiments, n is an integer between 1 and 15, inclusive. In certain embodiments, n is an integer between 1 and 10, inclusive. In certain embodiments, n is an integer between 1 and 5, inclusive. In certain embodiments, the molecule is of the formula:

wherein X, R¹, R², and R³ are as defined herein; and A′ is substituted or unsubstituted, cyclic or acyclic, branched or unbranched aliphatic; or substituted or unsubstituted, cyclic or acyclic, branched or unbranched heteroaliphatic. In certain embodiments, A′ is of one of the formulae:

In certain embodiments, A is of one of the formulae:

In certain embodiments, A is of one of the formulae:

In certain embodiments, A is of the formula:

In certain embodiments, A is of the formula:

In certain embodiments, R¹ is a steroid. In certain embodiments, R¹ is a cholesterol. In certain embodiments, R¹ is a lipophilic vitamin. In certain embodiments, R¹ is a vitamin A. In certain embodiments, R¹ is a vitamin E. In certain embodiments, R¹ is of the formula:

wherein R^(A) is substituted or unsubstituted, cyclic or acyclic, branched or unbranched aliphatic; or substituted or unsubstituted, cyclic or acyclic, branched or unbranched heteroaliphatic. In certain embodiments, R¹ is of the formula:

In certain embodiments, R¹ is of the formula:

In certain embodiments, R¹ is of the formula:

In certain embodiments, R¹ is of the formula:

In certain embodiments, R¹ is of the formula:

In certain embodiments, the nucleic acid molecule is of the formula:

wherein X is N or CH; A is a bond; substituted or unsubstituted, cyclic or acyclic, branched or unbranched aliphatic; or substituted or unsubstituted, cyclic or acyclic, branched or unbranched heteroaliphatic; R¹ is a hydrophobic moiety; R² is hydrogen; an oxygen-protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; and R³ is a nucleic acid. In certain embodiments, the nucleic acid molecule is of the formula:

wherein X is N or CH; A is a bond; substituted or unsubstituted, cyclic or acyclic, branched or unbranched aliphatic; or substituted or unsubstituted, cyclic or acyclic, branched or unbranched heteroaliphatic; R¹ is a hydrophobic moiety; R² is hydrogen: an oxygen-protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; and R³ is a nucleic acid. In certain embodiments, the nucleic acid molecule is of the formula:

wherein X is N or CH; A is a bond; substituted or unsubstituted, cyclic or acyclic, branched or unbranched aliphatic; or substituted or unsubstituted, cyclic or acyclic, branched or unbranched heteroaliphatic; R¹ is a hydrophobic moiety; R² is hydrogen; an oxygen-protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; and R³ is a nucleic acid. In certain embodiments, the nucleic acid molecule is of the formula:

In certain embodiments, the nucleic acid molecule is of the formula:

In certain embodiments, the nucleic acid molecule is of the formula:

wherein R³ is a nucleic acid. In certain embodiments, the nucleic acid molecule is of the formula:

wherein R³ is a nucleic acid; and n is an integer between 1 and 20, inclusive. In certain embodiments, the nucleic acid molecule is of the formula:

In certain embodiments, the nucleic acid molecule is of the formula:

In certain embodiments, the nucleic acid molecule is of the formula:

In certain embodiments, the nucleic acid molecule is of the formula:

In certain embodiments, the nucleic acid molecule is of the formula:

As used herein, the term “linkage” includes a naturally occurring, unmodified phosphodiester moiety (—O—(PO²⁻)—O—) that covalently couples adjacent nucleomonomers. As used herein, the term “substitute linkage” includes any analog or derivative of the native phosphodiester group that covalently couples adjacent nucleomonomers. Substitute linkages include phosphodiester analogs, e.g., phosphorothioate, phosphorodithioate, and P-ethyoxyphosphodiester, P-ethoxyphosphodiester, P-alkyloxyphosphotriester, methylphosphonate, and nonphosphorus containing linkages, e.g., acetals and amides. Such substitute linkages are known in the art (e.g., Bjergarde et al. 1991. Nucleic Acids Res. 19:5843; Caruthers et al. 1991. Nucleosides Nucleotides. 10:47). In certain embodiments, non-hydrolizable linkages are preferred, such as phosphorothioate linkages.

In certain embodiments, oligonucleotides of the invention comprise hydrophobicly modified nucleotides or “hydrophobic modifications.” As used herein “hydrophobic modifications” refers to bases that are modified such that (1) overall hydrophobicity of the base is significantly increased, and/or (2) the base is still capable of forming close to regular Watson-Crick interaction. Several non-limiting examples of base modifications include 5-position uridine and cytidine modifications such as phenyl, 4-pyridyl, 2-pyridyl, indolyl, and isobutyl, phenyl (C6H5OH); tryptophanyl (C8H6N)CH2CH(NH2)CO), Isobutyl, butyl, aminobenzyl; phenyl; and naphthyl.

Another type of conjugates that can be attached to the end (3′ or 5′ end), the loop region, or any other parts of the sd-rxRNA might include a sterol, sterol type molecule, peptide, small molecule, protein, etc. In some embodiments, a sd-rxRNA may contain more than one conjugates (same or different chemical nature). In some embodiments, the conjugate is cholesterol.

Another way to increase target gene specificity, or to reduce off-target silencing effect, is to introduce a 2′-modification (such as the 2′-O methyl modification) at a position corresponding to the second 5′-end nucleotide of the guide sequence. This allows the positioning of this 2′-modification in the Dicer-resistant hairpin structure, thus enabling one to design better RNAi constructs with less or no off-target silencing.

In one embodiment, a hairpin polynucleotide of the invention can comprise one nucleic acid portion which is DNA and one nucleic acid portion which is RNA. Antisense (guide) sequences of the invention can be “chimeric oligonucleotides” which comprise an RNA-like and a DNA-like region.

The language “RNase H activating region” includes a region of an oligonucleotide. e.g., a chimeric oligonucleotide, that is capable of recruiting RNase H to cleave the target RNA strand to which the oligonucleotide binds. Typically, the RNase activating region contains a minimal core (of at least about 3-5, typically between about 3-12, more typically, between about 5-12, and more preferably between about 5-10 contiguous nucleomonomers) of DNA or DNA-like nucleomonomers. (See, e.g., U.S. Pat. No. 5,849,902). Preferably, the RNase H activating region comprises about nine contiguous deoxyribose containing nucleomonomers.

The language “non-activating region” includes a region of an antisense sequence, e.g., a chimeric oligonucleotide, that does not recruit or activate RNase H. Preferably, a non-activating region does not comprise phosphorothioate DNA. The oligonucleotides of the invention comprise at least one non-activating region. In one embodiment, the non-activating region can be stabilized against nucleases or can provide specificity for the target by being complementary to the target and forming hydrogen bonds with the target nucleic acid molecule, which is to be bound by the oligonucleotide.

In one embodiment, at least a portion of the contiguous polynucleotides are linked by a substitute linkage, e.g., a phosphorothioate linkage.

In certain embodiments, most or all of the nucleotides beyond the guide sequence (2′-modified or not) are linked by phosphorothioate linkages. Such constructs tend to have improved pharmacokinetics due to their higher affinity for serum proteins. The phosphorothioate linkages in the non-guide sequence portion of the polynucleotide generally do not interfere with guide strand activity, once the latter is loaded into RISC.

Antisense (guide) sequences of the present invention may include “morpholino oligonucleotides.” Morpholino oligonucleotides are non-ionic and function by an RNase H-independent mechanism. Each of the 4 genetic bases (Adenine, Cytosine, Guanine, and Thymine/Uracil) of the morpholino oligonucleotides is linked to a 6-membered morpholine ring. Morpholino oligonucleotides are made by joining the 4 different subunit types by, e.g., non-ionic phosphorodiamidate inter-subunit linkages. Morpholino oligonucleotides have many advantages including: complete resistance to nucleases (Antisense & Nucl. Acid Drug Dev. 1996. 6:267); predictable targeting (Biochemica Biophysica Acta. 1999. 1489:141); reliable activity in cells (Antisense & Nucl. Acid Drug Dev. 1997. 7:63); excellent sequence specificity (Antisense & Nucl. Acid Drug Dev. 1997. 7:151); minimal non-antisense activity (Biochemica Biophysica Acta. 1999. 1489:141); and simple osmotic or scrape delivery (Antisense & Nucl. Acid Drug Dev. 1997. 7:291). Morpholino oligonucleotides are also preferred because of their non-toxicity at high doses. A discussion of the preparation of morpholino oligonucleotides can be found in Antisense & Nucl. Acid Drug Dev. 1997. 7:187.

The chemical modifications described herein are believed, based on the data described herein, to promote single stranded polynucleotide loading into the RISC. Single stranded polynucleotides have been shown to be active in loading into RISC and inducing gene silencing. However, the level of activity for single stranded polynucleotides appears to be 2 to 4 orders of magnitude lower when compared to a duplex polynucleotide.

The present invention provides a description of the chemical modification patterns, which may (a) significantly increase stability of the single stranded polynucleotide (b) promote efficient loading of the polynucleotide into the RISC complex and (c) improve uptake of the single stranded nucleotide by the cell. FIG. 5 provides some non-limiting examples of the chemical modification patterns which may be beneficial for achieving single stranded polynucleotide efficacy inside the cell. The chemical modification patterns may include combination of ribose, backbone, hydrophobic nucleoside and conjugate type of modifications. In addition, in some of the embodiments, the 5′ end of the single polynucleotide may be chemically phosphorylated.

In yet another embodiment, the present invention provides a description of the chemical modifications patterns, which improve functionality of RISC inhibiting polynucleotides. Single stranded polynucleotides have been shown to inhibit activity of a preloaded RISC complex through the substrate competition mechanism. For these types of molecules, conventionally called antagomers, the activity usually requires high concentration and in vivo delivery is not very effective. The present invention provides a description of the chemical modification patterns, which may (a) significantly increase stability of the single stranded polynucleotide (b) promote efficient recognition of the polynucleotide by the RISC as a substrate and/or (c) improve uptake of the single stranded nucleotide by the cell. FIG. 6 provides some non-limiting examples of the chemical modification patterns that may be beneficial for achieving single stranded polynucleotide efficacy inside the cell. The chemical modification patterns may include combination of ribose, backbone, hydrophobic nucleoside and conjugate type of modifications.

The modifications provided by the present invention are applicable to all polynucleotides. This includes single stranded RISC entering polynucleotides, single stranded RISC inhibiting polynucleotides, conventional duplexed polynucleotides of variable length (15-40 bp),asymmetric duplexed polynucleotides, and the like. Polynucleotides may be modified with wide variety of chemical modification patterns, including κ′ end, ribose, backbone and hydrophobic nucleoside modifications.

Synthesis

Oligonucleotides of the invention can be synthesized by any method known in the art, e.g., using enzymatic synthesis and/or chemical synthesis. The oligonucleotides can be synthesized in vitro (e.g., using enzymatic synthesis and chemical synthesis) or in vivo (using recombinant DNA technology well known in the art).

In a preferred embodiment, chemical synthesis is used for modified polynucleotides. Chemical synthesis of linear oligonucleotides is well known in the art and can be achieved by solution or solid phase techniques. Preferably, synthesis is by solid phase methods. Oligonucleotides can be made by any of several different synthetic procedures including the phosphoramidite, phosphite triester, H-phosphonate, and phosphotriester methods, typically by automated synthesis methods.

Oligonucleotide synthesis protocols are well known in the art and can be found, e.g., in U.S. Pat. No. 5,830,653; WO 98/13526; Stec et al. 1984. J. Am. Chem. Soc. 106:6077; Stec et al. 1985. J. Org. Chem. 50:3908; Stec et al. J. Chromatog. 1985. 326:263; LaPlanche et al. 1986. Nucl. Acid. Res. 1986. 14:9081; Fasman G. D., 1989. Practical Handbook of Biochemistry and Molecular Biology. 1989. CRC Press, Boca Raton, Fla.; Lamone. 1993. Biochem. Soc. Trans. 21:1; U.S. Pat. Nos. 5,013,830; 5,214,135; 5,525,719; Kawasaki et al. 1993. J. Med. Chem. 36:831; WO 92/03568; U.S. Pat. Nos. 5,276,019; and 5,264,423.

The synthesis method selected can depend on the length of the desired oligonucleotide and such choice is within the skill of the ordinary artisan. For example, the phosphoramidite and phosphite triester method can produce oligonucleotides having 175 or more nucleotides, while the H-phosphonate method works well for oligonucleotides of less than 100 nucleotides. If modified bases are incorporated into the oligonucleotide, and particularly if modified phosphodiester linkages are used, then the synthetic procedures are altered as needed according to known procedures. In this regard, Uhlmann et al. (1990, Chemical Reviews 90:543-584) provide references and outline procedures for making oligonucleotides with modified bases and modified phosphodiester linkages. Other exemplary methods for making oligonucleotides are taught in Sonveaux. 1994. “Protecting Groups in Oligonucleotide Synthesis”; Agrawal. Methods in Molecular Biology 26:1. Exemplary synthesis methods are also taught in “Oligonucleotide Synthesis—A Practical Approach” (Gait, M. J. IRL Press at Oxford University Press. 1984). Moreover, linear oligonucleotides of defined sequence, including some sequences with modified nucleotides, are readily available from several commercial sources.

The oligonucleotides may be purified by polyacrylamide gel electrophoresis, or by any of a number of chromatographic methods, including gel chromatography and high pressure liquid chromatography. To confirm a nucleotide sequence, especially unmodified nucleotide sequences, oligonucleotides may be subjected to DNA sequencing by any of the known procedures, including Maxam and Gilbert sequencing, Sanger sequencing, capillary electrophoresis sequencing, the wandering spot sequencing procedure or by using selective chemical degradation of oligonucleotides bound to Hybond paper. Sequences of short oligonucleotides can also be analyzed by laser desorption mass spectroscopy or by fast atom bombardment (McNeal, et al., 1982, J. Am. Chem. Soc. 104:976; Viari, et al., 1987, Biomed. Environ. Mass Spectrom. 14:83; Grotjahn et al., 1982, Nuc. Acid Res. 10:4671). Sequencing methods are also available for RNA oligonucleotides.

The quality of oligonucleotides synthesized can be verified by testing the oligonucleotide by capillary electrophoresis and denaturing strong anion HPLC (SAX-HPLC) using, e.g., the method of Bergot and Egan. 1992. J. Chrom. 599:35.

Other exemplary synthesis techniques are well known in the art (see, e.g., Sambrook et al., Molecular Cloning: a Laboratory Manual. Second Edition (1989); DNA Cloning, Volumes I and II (DN Glover Ed. 1985); Oligonucleotide Synthesis (M J Gait Ed, 1984; Nucleic Acid Hybridisation (B D Hames and S J Higgins eds. 1984); A Practical Guide to Molecular Cloning (1984); or the series, Methods in Enzymology (Academic Press, Inc.)).

In certain embodiments, the subject RNAi constructs or at least portions thereof are transcribed from expression vectors encoding the subject constructs. Any art recognized vectors may be use for this purpose. The transcribed RNAi constructs may be isolated and purified, before desired modifications (such as replacing an unmodified sense strand with a modified one, etc.) are carried out.

Delivery/Carrier

Uptake of Oligonucleotides by Cells

Oligonucleotides and oligonucleotide compositions are contacted with (i.e., brought into contact with, also referred to herein as administered or delivered to) and taken up by one or more cells or a cell lysate. The term “cells” includes prokaryotic and eukaryotic cells, preferably vertebrate cells, and, more preferably, mammalian cells. In a preferred embodiment, the oligonucleotide compositions of the invention are contacted with human cells.

Oligonucleotide compositions of the invention can be contacted with cells in vitro, e.g., in a test tube or culture dish, (and may or may not be introduced into a subject) or in vivo. e.g., in a subject such as a mammalian subject. Oligonucleotides are taken up by cells at a slow rate by endocytosis, but endocytosed oligonucleotides are generally sequestered and not available, e.g., for hybridization to a target nucleic acid molecule. In one embodiment, cellular uptake can be facilitated by electroporation or calcium phosphate precipitation. However, these procedures are only useful for in vitro or ex vivo embodiments, are not convenient and, in some cases, are associated with cell toxicity.

In another embodiment, delivery of oligonucleotides into cells can be enhanced by suitable art recognized methods including calcium phosphate. DMSO, glycerol or dextran, electroporation, or by transfection, e.g., using cationic, anionic, or neutral lipid compositions or liposomes using methods known in the art (see e.g., WO 90/14074; WO 91/16024; WO 91/17424; U.S. Pat. No. 4,897,355; Bergan et al. 1993. Nucleic Acids Research. 21:3567). Enhanced delivery of oligonucleotides can also be mediated by the use of vectors (See e.g., Shi, Y. 2003. Trends Genet 2003 Jan. 19:9; Reichhart J M et al. Genesis. 2002. 34(1-2): 1604, Yu et al. 2002. Proc. Natl. Acad Sci. USA 99:6047; Sui et al. 2002. Proc. Natl. Acad Sci. USA 99:5515) viruses, polyamine or polycation conjugates using compounds such as polylysine, protamine, or Ni, N12-bis (ethyl) spermine (see, e.g., Bartzatt, R. et al. 1989. Biotechnol. Appl. Biochem. 11:133; Wagner E. et al. 1992. Proc. Natl. Acad. Sci. 88:4255).

In certain embodiments, the sd-rxRNA of the invention may be delivered by using various beta-glucan containing particles, referred to as GeRPs (glucan encapsulated RNA loaded particle), described in, and incorporated by reference from, U.S. Provisional Application No. 61/310,611, filed on Mar. 4, 2010 and entitled “Formulations and Methods for Targeted Delivery to Phagocyte Cells.” Such particles are also described in, and incorporated by reference from US Patent Publications US 2005/0281781 A1, and US 2010/0040656, and in PCT publications WO 2006/007372, and WO 2007/050643. The sd-rxRNA molecule may be hydrophobically modified and optionally may be associated with a lipid and/or amphiphilic peptide. In certain embodiments, the beta-glucan particle is derived from yeast. In certain embodiments, the payload trapping molecule is a polymer, such as those with a molecular weight of at least about 1000 Da, 10,000 Da, 50.000 Da, 100 kDa, 500 kDa, etc. Preferred polymers include (without limitation) cationic polymers, chitosans, or PEI (polyethylenimine), etc.

Glucan particles can be derived from insoluble components of fungal cell walls such as yeast cell walls. In some embodiments, the yeast is Baker's yeast. Yeast-derived glucan molecules can include one or more of β-(1,3)-Glucan, β-(1,6)-Glucan, mannan and chitin. In some embodiments, a glucan particle comprises a hollow yeast cell wall whereby the particle maintains a three dimensional structure resembling a cell, within which it can complex with or encapsulate a molecule such as an RNA molecule. Some of the advantages associated with the use of yeast cell wall particles are availability of the components, their biodegradable nature, and their ability to be targeted to phagocytic cells.

In some embodiments, glucan particles can be prepared by extraction of insoluble components from cell walls, for example by extracting Baker's yeast (Fleischmann's) with 1M NaOH/pH 4.0 H2O, followed by washing and drying. Methods of preparing yeast cell wall particles are discussed in, and incorporated by reference from U.S. Pat. Nos. 4,810,646, 4,992,540, 5,082,936, 5,028,703, 5,032,401, 5,322,841, 5,401,727, 5,504,079, 5,607,677, 5,968,811, 6,242,594, 6,444,448, 6,476,003, US Patent Publications 2003/0216346, 2004/0014715 and 2010/0040656, and PCT published application WO02/12348.

Protocols for preparing glucan particles are also described in, and incorporated by reference from, the following references: Soto and Ostroff (2008), “Characterization of multilayered nanoparticles encapsulated in yeast cell wall particles for DNA delivery.” Bioconjug Chem 19(4):840-8; Soto and Ostroff (2007), “Oral Macrophage Mediated Gene Delivery System,” Nanotech, Volume 2, Chapter 5 (“Drug Delivery”), pages 378-381; and Li et al. (2007). “Yeast glucan particles activate murine resident macrophages to secrete proinflammatory cytokines via MyD88- and Syk kinase-dependent pathways.” Clinical Immunology 124(2):170-181.

Glucan containing particles such as yeast cell wall particles can also be obtained commercially. Several non-limiting examples include: Nutricell MOS 55 from Biorigin (Sao Paolo, Brazil), SAF-Mannan (SAF Agri, Minneapolis, Minn.), Nutrex (Sensient Technologies, Milwaukee, Wis.), alkali-extracted particles such as those produced by Nutricepts (Nutricepts Inc., Burnsville. Minn.) and ASA Biotech, acid-extracted WGP particles from Biopolymer Engineering, and organic solvent-extracted particles such as Adjuvax™ from Alpha-beta Technology, Inc. (Worcester, Mass.) and microparticulate glucan from Novogen (Stamford, Conn.).

Glucan particles such as yeast cell wall particles can have varying levels of purity depending on the method of production and/or extraction. In some instances, particles are alkali-extracted, acid-extracted or organic solvent-extracted to remove intracellular components and/or the outer mannoprotein layer of the cell wall. Such protocols can produce particles that have a glucan (w/w) content in the range of 50%-90%. In some instances, a particle of lower purity, meaning lower glucan w/w content may be preferred, while in other embodiments, a particle of higher purity, meaning higher glucan w/w content may be preferred.

Glucan particles, such as yeast cell wall particles, can have a natural lipid content. For example, the particles can contain 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% or more than 20% w/w lipid. In the Examples section, the effectiveness of two glucan particle batches are tested: YGP SAF and YGP SAF+L (containing natural lipids). In some instances, the presence of natural lipids may assist in complexation or capture of RNA molecules.

Glucan containing particles typically have a diameter of approximately 2-4 microns, although particles with a diameter of less than 2 microns or greater than 4 microns are also compatible with aspects of the invention.

The RNA molecule(s) to be delivered are complexed or “trapped” within the shell of the glucan particle. The shell or RNA component of the particle can be labeled for visualization, as described in, and incorporated by reference from, Soto and Ostroff (2008) Bioconjug Chem 19:840. Methods of loading GeRPs are discussed further below.

The optimal protocol for uptake of oligonucleotides will depend upon a number of factors, the most crucial being the type of cells that are being used. Other factors that are important in uptake include, but are not limited to, the nature and concentration of the oligonucleotide, the confluence of the cells, the type of culture the cells are in (e.g., a suspension culture or plated) and the type of media in which the cells are grown.

Encapsulating Agents

Encapsulating agents entrap oligonucleotides within vesicles. In another embodiment of the invention, an oligonucleotide may be associated with a carrier or vehicle, e.g., liposomes or micelles, although other carriers could be used, as would be appreciated by one skilled in the art. Liposomes are vesicles made of a lipid bilayer having a structure similar to biological membranes. Such carriers are used to facilitate the cellular uptake or targeting of the oligonucleotide, or improve the oligonucleotides pharmacokinetic or toxicological properties.

For example, the oligonucleotides of the present invention may also be administered encapsulated in liposomes, pharmaceutical compositions wherein the active ingredient is contained either dispersed or variously present in corpuscles consisting of aqueous concentric layers adherent to lipidic layers. The oligonucleotides, depending upon solubility, may be present both in the aqueous layer and in the lipidic layer, or in what is generally termed a liposomic suspension. The hydrophobic layer, generally but not exclusively, comprises phopholipids such as lecithin and sphingomyelin, steroids such as cholesterol, more or less ionic surfactants such as diacetylphosphate, stearylamine, or phosphatidic acid, or other materials of a hydrophobic nature. The diameters of the liposomes generally range from about 15 nm to about 5 microns.

The use of liposomes as drug delivery vehicles offers several advantages. Liposomes increase intracellular stability, increase uptake efficiency and improve biological activity. Liposomes are hollow spherical vesicles composed of lipids arranged in a similar fashion as those lipids which make up the cell membrane. They have an internal aqueous space for entrapping water soluble compounds and range in size from 0.05 to several microns in diameter. Several studies have shown that liposomes can deliver nucleic acids to cells and that the nucleic acids remain biologically active. For example, a lipid delivery vehicle originally designed as a research tool, such as Lipofectin or LIPOFECTAMINE™ 2000, can deliver intact nucleic acid molecules to cells.

Specific advantages of using liposomes include the following: they are non-toxic and biodegradable in composition; they display long circulation half-lives; and recognition molecules can be readily attached to their surface for targeting to tissues. Finally, cost-effective manufacture of liposome-based pharmaceuticals, either in a liquid suspension or lyophilized product, has demonstrated the viability of this technology as an acceptable drug delivery system.

In some aspects, formulations associated with the invention might be selected for a class of naturally occurring or chemically synthesized or modified saturated and unsaturated fatty acid residues. Fatty acids might exist in a form of triglycerides, diglycerides or individual fatty acids. In another embodiment, the use of well-validated mixtures of fatty acids and/or fat emulsions currently used in pharmacology for parenteral nutrition may be utilized.

Liposome based formulations are widely used for oligonucleotide delivery. However, most of commercially available lipid or liposome formulations contain at least one positively charged lipid (cationic lipids). The presence of this positively charged lipid is believed to be essential for obtaining a high degree of oligonucleotide loading and for enhancing liposome fusogenic properties. Several methods have been performed and published to identify optimal positively charged lipid chemistries. However, the commercially available liposome formulations containing cationic lipids are characterized by a high level of toxicity. In vivo limited therapeutic indexes have revealed that liposome formulations containing positive charged lipids are associated with toxicity (i.e. elevation in liver enzymes) at concentrations only slightly higher than concentration required to achieve RNA silencing.

Nucleic acids associated with the invention can be hydrophobically modified and can be encompassed within neutral nanotransporters. Further description of neutral nanotransporters is incorporated by reference from PCT Application PCT/US2009/005251, filed on Sep. 22, 2009, and entitled “Neutral Nanotransporters.” Such particles enable quantitative oligonucleotide incorporation into non-charged lipid mixtures. The lack of toxic levels of cationic lipids in such neutral nanotransporter compositions is an important feature.

As demonstrated in PCT/US2009/005251, oligonucleotides can effectively be incorporated into a lipid mixture that is free of cationic lipids and such a composition can effectively deliver a therapeutic oligonucleotide to a cell in a manner that it is functional. For example, a high level of activity was observed when the fatty mixture was composed of a phosphatidylcholine base fatty acid and a sterol such as a cholesterol. For instance, one preferred formulation of neutral fatty mixture is composed of at least 20% of DOPC or DSPC and at least 20% of sterol such as cholesterol. Even as low as 1:5 lipid to oligonucleotide ratio was shown to be sufficient to get complete encapsulation of the oligonucleotide in a non charged formulation.

The neutral nanotransporters compositions enable efficient loading of oligonucleotide into neutral fat formulation. The composition includes an oligonucleotide that is modified in a manner such that the hydrophobicity of the molecule is increased (for example a hydrophobic molecule is attached (covalently or no-covalently) to a hydrophobic molecule on the oligonucleotide terminus or a non-terminal nucleotide, base, sugar, or backbone), the modified oligonucleotide being mixed with a neutral fat formulation (for example containing at least 25% of cholesterol and 25% of DOPC or analogs thereof). A cargo molecule, such as another lipid can also be included in the composition. This composition, where part of the formulation is build into the oligonucleotide itself, enables efficient encapsulation of oligonucleotide in neutral lipid particles.

In some aspects, stable particles ranging in size from 50 to 140 nm can be formed upon complexing of hydrophobic oligonucleotides with preferred formulations. It is interesting to mention that the formulation by itself typically does not form small particles, but rather, forms agglomerates, which are transformed into stable 50-120 nm particles upon addition of the hydrophobic modified oligonucleotide.

The neutral nanotransporter compositions of the invention include a hydrophobic modified polynucleotide, a neutral fatty mixture, and optionally a cargo molecule. A “hydrophobic modified polynucleotide” as used herein is a polynucleotide of the invention (i.e. sd-rxRNA) that has at least one modification that renders the polynucleotide more hydrophobic than the polynucleotide was prior to modification. The modification may be achieved by attaching (covalently or non-covalently) a hydrophobic molecule to the polynucleotide. In some instances the hydrophobic molecule is or includes a lipophilic group.

The term “lipophilic group” means a group that has a higher affinity for lipids than its affinity for water. Examples of lipophilic groups include, but are not limited to, cholesterol, a cholesteryl or modified cholesteryl residue, adamantine, dihydrotesterone, long chain alkyl, long chain alkenyl, long chain alkynyl, olely-lithocholic, cholenic, oleoyl-cholenic, palmityl, heptadecyl, myrisityl, bile acids, cholic acid or taurocholic acid, deoxycholate, oleyl litocholic acid, oleoyl cholenic acid, glycolipids, phospholipids, sphingolipids, isoprenoids, such as steroids, vitamins, such as vitamin E, fatty acids either saturated or unsaturated, fatty acid esters, such as triglycerides, pyrenes, porphyrines. Texaphyrine, adamantane, acridines, biotin, coumarin, fluorescein, rhodamine, Texas-Red, digoxygenin, dimethoxytrityl, t-butyldimethylsilyl, t-butyldiphenylsilyl, cyanine dyes (e.g. Cy3 or Cy5), Hoechst 33258 dye, psoralen, or ibuprofen. The cholesterol moiety may be reduced (e.g. as in cholestan) or may be substituted (e.g. by halogen). A combination of different lipophilic groups in one molecule is also possible.

The hydrophobic molecule may be attached at various positions of the polynucleotide. As described above, the hydrophobic molecule may be linked to the terminal residue of the polynucleotide such as the 3′ of 5′-end of the polynucleotide. Alternatively, it may be linked to an internal nucleotide or a nucleotide on a branch of the polynucleotide. The hydrophobic molecule may be attached, for instance to a 2′-position of the nucleotide. The hydrophobic molecule may also be linked to the heterocyclic base, the sugar or the backbone of a nucleotide of the polynucleotide.

The hydrophobic molecule may be connected to the polynucleotide by a linker moiety. Optionally the linker moiety is a non-nucleotidic linker moiety. Non-nucleotidic linkers are e.g. abasic residues (dSpacer), oligoethyleneglycol, such as triethyleneglycol (spacer 9) or hexaethylenegylcol (spacer 18), or alkane-diol, such as butanediol. The spacer units are preferably linked by phosphodiester or phosphorothioate bonds. The linker units may appear just once in the molecule or may be incorporated several times. e.g. via phosphodiester, phosphorothioate, methylphosphonate, or amide linkages.

Typical conjugation protocols involve the synthesis of polynucleotides bearing an aminolinker at one or more positions of the sequence, however, a linker is not required. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the polynucleotide still bound to a solid support or following cleavage of the polynucleotide in solution phase. Purification of the modified polynucleotide by HPLC typically results in a pure material.

In some embodiments the hydrophobic molecule is a sterol type conjugate, a PhytoSterol conjugate, cholesterol conjugate, sterol type conjugate with altered side chain length, fatty acid conjugate, any other hydrophobic group conjugate, and/or hydrophobic modifications of the internal nucleoside, which provide sufficient hydrophobicity to be incorporated into micelles.

For purposes of the present invention, the term “sterols”, refers or steroid alcohols are a subgroup of steroids with a hydroxyl group at the 3-position of the A-ring. They are amphipathic lipids synthesized from acetyl-coenzyme A via the HMG-CoA reductase pathway. The overall molecule is quite flat. The hydroxyl group on the A ring is polar. The rest of the aliphatic chain is non-polar. Usually sterols are considered to have an 8 carbon chain at position 17.

For purposes of the present invention, the term “sterol type molecules”, refers to steroid alcohols, which are similar in structure to sterols. The main difference is the structure of the ring and number of carbons in a position 21 attached side chain.

For purposes of the present invention, the term “PhytoSterols” (also called plant sterols) are a group of steroid alcohols, phytochemicals naturally occurring in plants. There are more then 200 different known PhytoSterols

For purposes of the present invention, the term “Sterol side chain” refers to a chemical composition of a side chain attached at the position 17 of sterol-type molecule. In a standard definition sterols are limited to a 4 ring structure carrying a 8 carbon chain at position 17. In this invention, the sterol type molecules with side chain longer and shorter than conventional are described. The side chain may branched or contain double back bones.

Thus, sterols useful in the invention, for example, include cholesterols, as well as unique sterols in which position 17 has attached side chain of 2-7 or longer then 9 carbons. In a particular embodiment, the length of the polycarbon tail is varied between and 9 carbons. Such conjugates may have significantly better in vivo efficacy, in particular delivery to liver. These types of molecules are expected to work at concentrations 5 to 9 fold lower then oligonucleotides conjugated to conventional cholesterols.

Alternatively the polynucleotide may be bound to a protein, peptide or positively charged chemical that functions as the hydrophobic molecule. The proteins may be selected from the group consisting of protamine, dsRNA binding domain, and arginine rich peptides. Exemplary positively charged chemicals include spermine, spermidine, cadaverine, and putrescine.

In another embodiment hydrophobic molecule conjugates may demonstrate even higher efficacy when it is combined with optimal chemical modification patterns of the polynucleotide (as described herein in detail), containing but not limited to hydrophobic modifications, phosphorothioate modifications, and 2′ ribo modifications.

In another embodiment the sterol type molecule may be a naturally occurring PhytoSterols. The polycarbon chain may be longer than 9 and may be linear, branched and/or contain double bonds. Some PhytoSterol containing polynucleotide conjugates may be significantly more potent and active in delivery of polynucleotides to various tissues. Some PhytoSterols may demonstrate tissue preference and thus be used as a way to delivery RNAi specifically to particular tissues.

The hydrophobic modified polynucleotide is mixed with a neutral fatty mixture to form a micelle. The neutral fatty acid mixture is a mixture of fats that has a net neutral or slightly net negative charge at or around physiological pH that can form a micelle with the hydrophobic modified polynucleotide. For purposes of the present invention, the term “micelle” refers to a small nanoparticle formed by a mixture of non charged fatty acids and phospholipids. The neutral fatty mixture may include cationic lipids as long as they are present in an amount that does not cause toxicity. In preferred embodiments the neutral fatty mixture is free of cationic lipids. A mixture that is free of cationic lipids is one that has less than 1% and preferably 0% of the total lipid being cationic lipid. The term “cationic lipid” includes lipids and synthetic lipids having a net positive charge at or around physiological pH. The term “anionic lipid” includes lipids and synthetic lipids having a net negative charge at or around physiological pH.

The neutral fats bind to the oligonucleotides of the invention by a strong but non-covalent attraction (e.g., an electrostatic, van der Waals, pi-stacking, etc. interaction).

The neutral fat mixture may include formulations selected from a class of naturally occurring or chemically synthesized or modified saturated and unsaturated fatty acid residues. Fatty acids might exist in a form of triglycerides, diglycerides or individual fatty acids. In another embodiment the use of well-validated mixtures of fatty acids and/or fat emulsions currently used in pharmacology for parenteral nutrition may be utilized.

The neutral fatty mixture is preferably a mixture of a choline based fatty acid and a sterol. Choline based fatty acids include for instance, synthetic phosphocholine derivatives such as DDPC, DLPC, DMPC, DPPC, DSPC, DOPC, POPC, and DEPC. DOPC (chemical registry number 4235-95-4) is dioleoylphosphatidylcholine (also known as dielaidoylphosphatidylcholine, dioleoyl-PC, dioleoylphosphocholine, dioleoyl-sn-glycero-3-phosphocholine, dioleylphosphatidylcholine). DSPC (chemical registry number 816-94-4) is distearoylphosphatidylcholine (also known as 1,2-Distearoyl-sn-Glycero-3-phosphocholine).

The sterol in the neutral fatty mixture may be for instance cholesterol. The neutral fatty mixture may be made up completely of a choline based fatty acid and a sterol or it may optionally include a cargo molecule. For instance, the neutral fatty mixture may have at least 20% or 25% fatty acid and 20% or 25% sterol.

For purposes of the present invention, the term “Fatty acids” relates to conventional description of fatty acid. They may exist as individual entities or in a form of two- and triglycerides. For purposes of the present invention, the term “fat emulsions” refers to safe fat formulations given intravenously to subjects who are unable to get enough fat in their diet. It is an emulsion of soy bean oil (or other naturally occurring oils) and egg phospholipids. Fat emulsions are being used for formulation of some insoluble anesthetics. In this disclosure, fat emulsions might be part of commercially available preparations like Intralipid, Liposyn. Nutrilipid, modified commercial preparations, where they are enriched with particular fatty acids or fully de novo-formulated combinations of fatty acids and phospholipids.

In one embodiment, the cells to be contacted with an oligonucleotide composition of the invention are contacted with a mixture comprising the oligonucleotide and a mixture comprising a lipid. e.g., one of the lipids or lipid compositions described supra for between about 12 hours to about 24 hours. In another embodiment, the cells to be contacted with an oligonucleotide composition are contacted with a mixture comprising the oligonucleotide and a mixture comprising a lipid, e.g., one of the lipids or lipid compositions described supra for between about 1 and about five days. In one embodiment, the cells are contacted with a mixture comprising a lipid and the oligonucleotide for between about three days to as long as about 30 days. In another embodiment, a mixture comprising a lipid is left in contact with the cells for at least about five to about 20 days. In another embodiment, a mixture comprising a lipid is left in contact with the cells for at least about seven to about 15 days.

50%-60% of the formulation can optionally be any other lipid or molecule. Such a lipid or molecule is referred to herein as a cargo lipid or cargo molecule. Cargo molecules include but are not limited to intralipid, small molecules, fusogenic peptides or lipids or other small molecules might be added to alter cellular uptake, endosomal release or tissue distribution properties. The ability to tolerate cargo molecules is important for modulation of properties of these particles, if such properties are desirable. For instance the presence of some tissue specific metabolites might drastically alter tissue distribution profiles. For example use of Intralipid type formulation enriched in shorter or longer fatty chains with various degrees of saturation affects tissue distribution profiles of these type of formulations (and their loads).

An example of a cargo lipid useful according to the invention is a fusogenic lipid. For instance, the zwitterionic lipid DOPE (chemical registry number 4004-5-1, 1,2-Dioleoyl-sn-Glycero-3-phosphoethanolamine) is a preferred cargo lipid.

Intralipid may be comprised of the following composition: 1000 mL contain: purified soybean oil 90 g, purified egg phospholipids 12 g, glycerol anhydrous 22 g, water for injection q.s. ad 1000 mL. pH is adjusted with sodium hydroxide to pH approximately 8. Energy content/L: 4.6 MJ (190 kcal). Osmolality (approx.): 300 mOsm/kg water. In another embodiment fat emulsion is Liposyn that contains 5% safflower oil, 5% soybean oil, up to 1.2% egg phosphatides added as an emulsifier and 2.5% glycerin in water for injection. It may also contain sodium hydroxide for pH adjustment. pH 8.0 (6.0-9.0). Liposyn has an osmolarity of 276 m Osmol/liter (actual).

Variation in the identity, amounts and ratios of cargo lipids affects the cellular uptake and tissue distribution characteristics of these compounds. For example, the length of lipid tails and level of saturability will affect differential uptake to liver, lung, fat and cardiomyocytes. Addition of special hydrophobic molecules like vitamins or different forms of sterols can favor distribution to special tissues which are involved in the metabolism of particular compounds. Complexes are formed at different oligonucleotide concentrations, with higher concentrations favoring more efficient complex formation.

In another embodiment, the fat emulsion is based on a mixture of lipids. Such lipids may include natural compounds, chemically synthesized compounds, purified fatty acids or any other lipids. In yet another embodiment the composition of fat emulsion is entirely artificial. In a particular embodiment, the fat emulsion is more then 70% linoleic acid. In yet another particular embodiment the fat emulsion is at least 1% of cardiolipin. Linoleic acid (LA) is an unsaturated omega-6 fatty acid. It is a colorless liquid made of a carboxylic acid with an 18-carbon chain and two cis double bonds.

In yet another embodiment of the present invention, the alteration of the composition of the fat emulsion is used as a way to alter tissue distribution of hydrophobicly modified polynucleotides. This methodology provides for the specific delivery of the polynucleotides to particular tissues (FIG. 12).

In another embodiment the fat emulsions of the cargo molecule contain more then 70% of Linoleic acid (C18H32O2) and/or cardiolipin are used for specifically delivering RNAi to heart muscle.

Fat emulsions, like intralipid have been used before as a delivery formulation for some non-water soluble drugs (such as Propofol, re-formulated as Diprivan). Unique features of the present invention include (a) the concept of combining modified polynucleotides with the hydrophobic compound(s), so it can be incorporated in the fat micelles and (b) mixing it with the fat emulsions to provide a reversible carrier. After injection into a blood stream, micelles usually bind to serum proteins, including albumin, HDL, LDL and other. This binding is reversible and eventually the fat is absorbed by cells. The polynucleotide, incorporated as a part of the micelle will then be delivered closely to the surface of the cells. After that cellular uptake might be happening though variable mechanisms, including but not limited to sterol type delivery.

Complexing Agents

Complexing agents bind to the oligonucleotides of the invention by a strong but non-covalent attraction (e.g., an electrostatic, van der Waals, pi-stacking, etc. interaction). In one embodiment, oligonucleotides of the invention can be complexed with a complexing agent to increase cellular uptake of oligonucleotides. An example of a complexing agent includes cationic lipids. Cationic lipids can be used to deliver oligonucleotides to cells. However, as discussed above, formulations free in cationic lipids are preferred in some embodiments.

The term “cationic lipid” includes lipids and synthetic lipids having both polar and non-polar domains and which are capable of being positively charged at or around physiological pH and which bind to polyanions, such as nucleic acids, and facilitate the delivery of nucleic acids into cells. In general cationic lipids include saturated and unsaturated alkyl and alicyclic ethers and esters of amines, amides, or derivatives thereof. Straight-chain and branched alkyl and alkenyl groups of cationic lipids can contain, e.g., from 1 to about 25 carbon atoms. Preferred straight chain or branched alkyl or alkene groups have six or more carbon atoms. Alicyclic groups include cholesterol and other steroid groups. Cationic lipids can be prepared with a variety of counterions (anions) including, e.g., Cl⁻, Br⁻, I⁻, F⁻, acetate, trifluoroacetate, sulfate, nitrite, and nitrate.

Examples of cationic lipids include polyethylenimine, polyamidoamine (PAMAM) starburst dendrimers, Lipofectin (a combination of DOTMA and DOPE), Lipofectase, LIPOFECTAMINE™ (e.g., LIPOFECTAMINE™ 2000), DOPE, Cytofectin (Gilead Sciences, Foster City, Calif.), and Eufectins (JBL. San Luis Obispo. Calif.). Exemplary cationic liposomes can be made from N-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA), N-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP), 3β-[N—(N′,N′-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol), 2,3,-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide; and dimethyldioctadecylammonium bromide (DDAB). The cationic lipid N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), for example, was found to increase 1000-fold the antisense effect of a phosphorothioate oligonucleotide. (Vlassov et al., 1994, Biochimica et Biophysica Acta 1197:95-108). Oligonucleotides can also be complexed with, e.g., poly (L-lysine) or avidin and lipids may, or may not, be included in this mixture, e.g., steryl-poly (L-lysine).

Cationic lipids have been used in the art to deliver oligonucleotides to cells (see, e.g., U.S. Pat. Nos. 5,855,910; 5,851,548; 5,830,430; 5,780,053; 5,767,099; Lewis et al. 1996. Proc. Natl. Acad. Sci. USA 93:3176; Hope et al. 1998. Molecular Membrane Biology 15:1). Other lipid compositions which can be used to facilitate uptake of the instant oligonucleotides can be used in connection with the claimed methods. In addition to those listed supra, other lipid compositions are also known in the art and include, e.g., those taught in U.S. Pat. Nos. 4,235,871; 4,501,728; 4,837,028; 4,737,323.

In one embodiment lipid compositions can further comprise agents, e.g., viral proteins to enhance lipid-mediated transfections of oligonucleotides (Kamata, et al., 1994. Nucl. Acids. Res. 22:536). In another embodiment, oligonucleotides are contacted with cells as part of a composition comprising an oligonucleotide, a peptide, and a lipid as taught, e.g., in U.S. Pat. No. 5,736,392. Improved lipids have also been described which are serum resistant (Lewis, et al., 1996. Proc. Natl. Acad. Sci. 93:3176). Cationic lipids and other complexing agents act to increase the number of oligonucleotides carried into the cell through endocytosis.

In another embodiment N-substituted glycine oligonucleotides (peptoids) can be used to optimize uptake of oligonucleotides. Peptoids have been used to create cationic lipid-like compounds for transfection (Murphy, et al., 1998. Proc. Natl. Acad. Sci. 95:1517). Peptoids can be synthesized using standard methods (e.g., Zuckermann, R. N., et al. 1992. J. Am. Chem. Soc. 114:10646; Zuckermann, R. N., et al. 1992. Int. J. Peptide Protein Res. 40:497). Combinations of cationic lipids and peptoids, liptoids, can also be used to optimize uptake of the subject oligonucleotides (Hunag, et al., 1998. Chemistry and Biology. 5:345). Liptoids can be synthesized by elaborating peptoid oligonucleotides and coupling the amino terminal submonomer to a lipid via its amino group (Hunag, et al., 1998. Chemistry and Biology. 5:345).

It is known in the art that positively charged amino acids can be used for creating highly active cationic lipids (Lewis et al. 1996. Proc. Natl. Acad. Sci. US.A. 93:3176). In one embodiment, a composition for delivering oligonucleotides of the invention comprises a number of arginine, lysine, histidine or ornithine residues linked to a lipophilic moiety (see e.g., U.S. Pat. No. 5,777,153).

In another embodiment, a composition for delivering oligonucleotides of the invention comprises a peptide having from between about one to about four basic residues. These basic residues can be located, e.g., on the amino terminal, C-terminal, or internal region of the peptide. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine (can also be considered non-polar), asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Apart from the basic amino acids, a majority or all of the other residues of the peptide can be selected from the non-basic amino acids, e.g., amino acids other than lysine, arginine, or histidine. Preferably a preponderance of neutral amino acids with long neutral side chains are used.

In one embodiment, a composition for delivering oligonucleotides of the invention comprises a natural or synthetic polypeptide having one or more gamma carboxyglutamic acid residues, or γ-Gla residues. These gamma carboxyglutamic acid residues may enable the polypeptide to bind to each other and to membrane surfaces. In other words, a polypeptide having a series of γ-Gla may be used as a general delivery modality that helps an RNAi construct to stick to whatever membrane to which it comes in contact. This may at least slow RNAi constructs from being cleared from the blood stream and enhance their chance of homing to the target.

The gamma carboxyglutamic acid residues may exist in natural proteins (for example, prothrombin has 10 γ-Gla residues). Alternatively, they can be introduced into the purified, recombinantly produced, or chemically synthesized polypeptides by carboxylation using, for example, a vitamin K-dependent carboxylase. The gamma carboxyglutamic acid residues may be consecutive or non-consecutive, and the total number and location of such gamma carboxyglutamic acid residues in the polypeptide can be regulated/fine tuned to achieve different levels of “stickiness” of the polypeptide.

In one embodiment, the cells to be contacted with an oligonucleotide composition of the invention are contacted with a mixture comprising the oligonucleotide and a mixture comprising a lipid. e.g., one of the lipids or lipid compositions described supra for between about 12 hours to about 24 hours. In another embodiment, the cells to be contacted with an oligonucleotide composition are contacted with a mixture comprising the oligonucleotide and a mixture comprising a lipid, e.g., one of the lipids or lipid compositions described supra for between about 1 and about five days. In one embodiment, the cells are contacted with a mixture comprising a lipid and the oligonucleotide for between about three days to as long as about 30 days. In another embodiment, a mixture comprising a lipid is left in contact with the cells for at least about five to about 20 days. In another embodiment, a mixture comprising a lipid is left in contact with the cells for at least about seven to about 15 days.

For example, in one embodiment, an oligonucleotide composition can be contacted with cells in the presence of a lipid such as cytofectin CS or GSV (available from Glen Research; Sterling, Va.), GS3815, GS2888 for prolonged incubation periods as described herein.

In one embodiment, the incubation of the cells with the mixture comprising a lipid and an oligonucleotide composition does not reduce the viability of the cells. Preferably, after the transfection period the cells are substantially viable. In one embodiment, after transfection, the cells are between at least about 70% and at least about 100% viable. In another embodiment, the cells are between at least about 80% and at least about 95% viable. In yet another embodiment, the cells are between at least about 85% and at least about 90% viable.

In one embodiment, oligonucleotides are modified by attaching a peptide sequence that transports the oligonucleotide into a cell, referred to herein as a “transporting peptide.” In one embodiment, the composition includes an oligonucleotide which is complementary to a target nucleic acid molecule encoding the protein, and a covalently attached transporting peptide.

The language “transporting peptide” includes an amino acid sequence that facilitates the transport of an oligonucleotide into a cell. Exemplary peptides which facilitate the transport of the moieties to which they are linked into cells are known in the art, and include, e.g., HIV TAT transcription factor, lactoferrin, Herpes VP22 protein, and fibroblast growth factor 2 (Pooga et al. 1998. Nature Biotechnology. 16:857; and Derossi et al. 1998. Trends in Cell Biology. 8:84; Elliott and O'Hare. 1997. Cell 88:223).

Oligonucleotides can be attached to the transporting peptide using known techniques, e.g., (Prochiantz. A. 1996. Curr. Opin. Neurobiol. 6:629; Derossi et al. 1998. Trends Cell Biol. 8:84; Troy et al. 1996. J. Neurosci. 16:253), Vives et al. 1997. J. Biol. Chem. 272:16010). For example, in one embodiment, oligonucleotides bearing an activated thiol group are linked via that thiol group to a cysteine present in a transport peptide (e.g., to the cysteine present in the β turn between the second and the third helix of the antennapedia homeodomain as taught, e.g., in Derossi et al. 1998. Trends Cell Biol. 8:84; Prochiantz. 1996. Current Opinion in Neurobiol. 6:629; Allinquant et al. 1995. J Cell Biol. 128:919). In another embodiment, a Boc-Cys-(Npys)OH group can be coupled to the transport peptide as the last (N-terminal) amino acid and an oligonucleotide bearing an SH group can be coupled to the peptide (Troy et al. 1996. J. Neurosci. 16:253).

In one embodiment, a linking group can be attached to a nucleomonomer and the transporting peptide can be covalently attached to the linker. In one embodiment, a linker can function as both an attachment site for a transporting peptide and can provide stability against nucleases. Examples of suitable linkers include substituted or unsubstituted C₁-C₂₀ alkyl chains, C₂-C₂₀ alkenyl chains. C₂-C₂₀ alkynyl chains, peptides, and heteroatoms (e.g., S, O, NH, etc.). Other exemplary linkers include bifunctional crosslinking agents such as sulfosuccinimidyl-4-(maleimidophenyl)-butyrate (SMPB) (see, e.g., Smith et al. Biochem J 1991. 276: 417-2).

In one embodiment, oligonucleotides of the invention are synthesized as molecular conjugates which utilize receptor-mediated endocytotic mechanisms for delivering genes into cells (see, e.g., Bunnell et al. 1992. Somatic Cell and Molecular Genetics. 18:559, and the references cited therein).

Targeting Agents

The delivery of oligonucleotides can also be improved by targeting the oligonucleotides to a cellular receptor. The targeting moieties can be conjugated to the oligonucleotides or attached to a carrier group (i.e., poly(L-lysine) or liposomes) linked to the oligonucleotides. This method is well suited to cells that display specific receptor-mediated endocytosis.

For instance, oligonucleotide conjugates to 6-phosphomannosylated proteins are internalized 20-fold more efficiently by cells expressing mannose 6-phosphate specific receptors than free oligonucleotides. The oligonucleotides may also be coupled to a ligand for a cellular receptor using a biodegradable linker. In another example, the delivery construct is mannosylated streptavidin which forms a tight complex with biotinylated oligonucleotides. Mannosylated streptavidin was found to increase 20-fold the internalization of biotinylated oligonucleotides. (Vlassov et al. 1994. Biochimica et Biophysica Acta 1197:95-108).

In addition specific ligands can be conjugated to the polylysine component of polylysine-based delivery systems. For example, transferrin-polylysine, adenovirus-polylysine, and influenza virus hemagglutinin HA-2 N-terminal fusogenic peptides-polylysine conjugates greatly enhance receptor-mediated DNA delivery in eucaryotic cells. Mannosylated glycoprotein conjugated to poly(L-lysine) in aveolar macrophages has been employed to enhance the cellular uptake of oligonucleotides. Liang et al. 1999. Pharmazie 54:559-566.

Because malignant cells have an increased need for essential nutrients such as folic acid and transferrin, these nutrients can be used to target oligonucleotides to cancerous cells. For example, when folic acid is linked to poly(L-lysine) enhanced oligonucleotide uptake is seen in promyelocytic leukaemia (HL-60) cells and human melanoma (M-14) cells. Ginobbi et al. 1997. Anticancer Res. 17:29. In another example, liposomes coated with maleylated bovine serum albumin, folic acid, or ferric protoporphyrin IX, show enhanced cellular uptake of oligonucleotides in murine macrophages, KB cells, and 2.2.15 human hepatoma cells. Liang et al. 1999. Pharmazie 54:559-566.

Liposomes naturally accumulate in the liver, spleen, and reticuloendothelial system (so-called, passive targeting). By coupling liposomes to various ligands such as antibodies are protein A, they can be actively targeted to specific cell populations. For example, protein A-bearing liposomes may be pretreated with H-2K specific antibodies which are targeted to the mouse major histocompatibility complex-encoded H-2K protein expressed on L cells. (Vlassov et al. 1994. Biochimica et Biophysica Acta 1197:95-108).

Other in vitro and/or in vivo delivery of RNAi reagents are known in the art, and can be used to deliver the subject RNAi constructs. See, for example, U.S. patent application publications 20080152661, 20080112916, 20080107694, 20080038296, 20070231392, 20060240093, 20060178327, 20060008910, 20050265957, 20050064595, 20050042227, 20050037496, 20050026286, 20040162235, 20040072785, 20040063654, 20030157030, WO 2008/036825, WO04/065601, and AU2004206255B2, just to name a few (all incorporated by reference).

Administration

The optimal course of administration or delivery of the oligonucleotides may vary depending upon the desired result and/or on the subject to be treated. As used herein “administration” refers to contacting cells with oligonucleotides and can be performed in vitro or in vivo. The dosage of oligonucleotides may be adjusted to optimally reduce expression of a protein translated from a target nucleic acid molecule, e.g., as measured by a readout of RNA stability or by a therapeutic response, without undue experimentation.

For example, expression of the protein encoded by the nucleic acid target can be measured to determine whether or not the dosage regimen needs to be adjusted accordingly. In addition, an increase or decrease in RNA or protein levels in a cell or produced by a cell can be measured using any art recognized technique. By determining whether transcription has been decreased, the effectiveness of the oligonucleotide in inducing the cleavage of a target RNA can be determined.

Any of the above-described oligonucleotide compositions can be used alone or in conjunction with a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” includes appropriate solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, it can be used in the therapeutic compositions. Supplementary active ingredients can also be incorporated into the compositions.

Oligonucleotides may be incorporated into liposomes or liposomes modified with polyethylene glycol or admixed with cationic lipids for parenteral administration. Incorporation of additional substances into the liposome, for example, antibodies reactive against membrane proteins found on specific target cells, can help target the oligonucleotides to specific cell types.

With respect to in vivo applications, the formulations of the present invention can be administered to a patient in a variety of forms adapted to the chosen route of administration, e.g., parenterally, orally, or intraperitoneally. Parenteral administration, which is preferred, includes administration by the following routes: intravenous; intramuscular; interstitially; intraarterially; subcutaneous; intra ocular; intrasynovial; trans epithelial, including transdermal; pulmonary via inhalation; ophthalmic; sublingual and buccal; topically, including ophthalmic; dermal; ocular, rectal; and nasal inhalation via insufflation. In preferred embodiments, the sd-rxRNA molecules are administered by intradermal injection or subcutaneously.

Pharmaceutical preparations for parenteral administration include aqueous solutions of the active compounds in water-soluble or water-dispersible form. In addition, suspensions of the active compounds as appropriate oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, or dextran, optionally, the suspension may also contain stabilizers. The oligonucleotides of the invention can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the oligonucleotides may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included in the invention.

Pharmaceutical preparations for topical administration include transdermal patches, ointments, lotions, creams, gels, drops, sprays, suppositories, liquids and powders. In addition, conventional pharmaceutical carriers, aqueous, powder or oily bases, or thickeners may be used in pharmaceutical preparations for topical administration.

Pharmaceutical preparations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. In addition, thickeners, flavoring agents, diluents, emulsifiers, dispersing aids, or binders may be used in pharmaceutical preparations for oral administration.

For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives, and detergents. Transmucosal administration may be through nasal sprays or using suppositories. For oral administration, the oligonucleotides are formulated into conventional oral administration forms such as capsules, tablets, and tonics. For topical administration, the oligonucleotides of the invention are formulated into ointments, salves, gels, or creams as known in the art.

Drug delivery vehicles can be chosen e.g., for in vitro, for systemic, or for topical administration. These vehicles can be designed to serve as a slow release reservoir or to deliver their contents directly to the target cell. An advantage of using some direct delivery drug vehicles is that multiple molecules are delivered per uptake. Such vehicles have been shown to increase the circulation half-life of drugs that would otherwise be rapidly cleared from the blood stream. Some examples of such specialized drug delivery vehicles which fall into this category are liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres.

The described oligonucleotides may be administered systemically to a subject. Systemic absorption refers to the entry of drugs into the blood stream followed by distribution throughout the entire body. Administration routes which lead to systemic absorption include: intravenous, subcutaneous, intraperitoneal, and intranasal. Each of these administration routes delivers the oligonucleotide to accessible diseased cells. Following subcutaneous administration, the therapeutic agent drains into local lymph nodes and proceeds through the lymphatic network into the circulation. The rate of entry into the circulation has been shown to be a function of molecular weight or size. The use of a liposome or other drug carrier localizes the oligonucleotide at the lymph node. The oligonucleotide can be modified to diffuse into the cell, or the liposome can directly participate in the delivery of either the unmodified or modified oligonucleotide into the cell.

The chosen method of delivery will result in entry into cells. In some embodiments, preferred delivery methods include liposomes (10-400 nm), hydrogels, controlled-release polymers, and other pharmaceutically applicable vehicles, and microinjection or electroporation (for ex vivo treatments).

The pharmaceutical preparations of the present invention may be prepared and formulated as emulsions. Emulsions are usually heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 pin in diameter. The emulsions of the present invention may contain excipients such as emulsifiers, stabilizers, dyes, fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives, and anti-oxidants may also be present in emulsions as needed. These excipients may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase.

Examples of naturally occurring emulsifiers that may be used in emulsion formulations of the present invention include lanolin, beeswax, phosphatides, lecithin and acacia. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. Examples of finely divided solids that may be used as emulsifiers include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

Examples of preservatives that may be included in the emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Examples of antioxidants that may be included in the emulsion formulations include free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

In one embodiment, the compositions of oligonucleotides are formulated as microemulsions. A microemulsion is a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution. Typically microemulsions are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a 4th component, generally an intermediate chain-length alcohol to form a transparent system.

Surfactants that may be used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (M0310), hexaglycerol monooleate (P0310), hexaglycerol pentaoleate (P0500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (S0750), decaglycerol decaoleate (DA0750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules.

Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C₈-C₁₂) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C₈-C₁₀ glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both oil/water and water/oil) have been proposed to enhance the oral bioavailability of drugs.

Microemulsions offer improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides el al., Pharmaceutical Research, 1994, 11:1385; Ho et al., J. Pharm. Sci., 1996, 85:138-143). Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of oligonucleotides from the gastrointestinal tract, as well as improve the local cellular uptake of oligonucleotides within the gastrointestinal tract, vagina, buccal cavity and other areas of administration.

In an embodiment, the present invention employs various penetration enhancers to affect the efficient delivery of nucleic acids, particularly oligonucleotides, to the skin of animals. Even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to increasing the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also act to enhance the permeability of lipophilic drugs.

Five categories of penetration enhancers that may be used in the present invention include: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants. Other agents may be utilized to enhance the penetration of the administered oligonucleotides include: glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-15 pyrrol, azones, and terpenes such as limonene, and menthone.

The oligonucleotides, especially in lipid formulations, can also be administered by coating a medical device, for example, a catheter, such as an angioplasty balloon catheter, with a cationic lipid formulation. Coating may be achieved, for example, by dipping the medical device into a lipid formulation or a mixture of a lipid formulation and a suitable solvent, for example, an aqueous-based buffer, an aqueous solvent, ethanol, methylene chloride, chloroform and the like. An amount of the formulation will naturally adhere to the surface of the device which is subsequently administered to a patient, as appropriate. Alternatively, a lyophilized mixture of a lipid formulation may be specifically bound to the surface of the device. Such binding techniques are described, for example, in K. Ishihara et al., Journal of Biomedical Materials Research, Vol. 27, pp. 1309-1314 (1993), the disclosures of which are incorporated herein by reference in their entirety.

The useful dosage to be administered and the particular mode of administration will vary depending upon such factors as the cell type, or for in vivo use, the age, weight and the particular animal and region thereof to be treated, the particular oligonucleotide and delivery method used, the therapeutic or diagnostic use contemplated, and the form of the formulation, for example, suspension, emulsion, micelle or liposome, as will be readily apparent to those skilled in the art. Typically, dosage is administered at lower levels and increased until the desired effect is achieved. When lipids are used to deliver the oligonucleotides, the amount of lipid compound that is administered can vary and generally depends upon the amount of oligonucleotide agent being administered. For example, the weight ratio of lipid compound to oligonucleotide agent is preferably from about 1:1 to about 15:1, with a weight ratio of about 5:1 to about 10:1 being more preferred. Generally, the amount of cationic lipid compound which is administered will vary from between about 0.1 milligram (mg) to about 1 gram (g). By way of general guidance, typically between about 0.1 mg and about 10 mg of the particular oligonucleotide agent, and about 1 mg to about 100 mg of the lipid compositions, each per kilogram of patient body weight, is administered, although higher and lower amounts can be used.

The agents of the invention are administered to subjects or contacted with cells in a biologically compatible form suitable for pharmaceutical administration. By “biologically compatible form suitable for administration” is meant that the oligonucleotide is administered in a form in which any toxic effects are outweighed by the therapeutic effects of the oligonucleotide. In one embodiment, oligonucleotides can be administered to subjects. Examples of subjects include mammals, e.g., humans and other primates; cows, pigs, horses, and farming (agricultural) animals; dogs, cats, and other domesticated pets; mice, rats, and transgenic non-human animals.

Administration of an active amount of an oligonucleotide of the present invention is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result. For example, an active amount of an oligonucleotide may vary according to factors such as the type of cell, the oligonucleotide used, and for in vivo uses the disease state, age, sex, and weight of the individual, and the ability of the oligonucleotide to elicit a desired response in the individual. Establishment of therapeutic levels of oligonucleotides within the cell is dependent upon the rates of uptake and efflux or degradation. Decreasing the degree of degradation prolongs the intracellular half-life of the oligonucleotide. Thus, chemically-modified oligonucleotides, e.g., with modification of the phosphate backbone, may require different dosing.

The exact dosage of an oligonucleotide and number of doses administered will depend upon the data generated experimentally and in clinical trials. Several factors such as the desired effect, the delivery vehicle, disease indication, and the route of administration, will affect the dosage. Dosages can be readily determined by one of ordinary skill in the art and formulated into the subject pharmaceutical compositions. Preferably, the duration of treatment will extend at least through the course of the disease symptoms.

Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, the oligonucleotide may be repeatedly administered, e.g., several doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. One of ordinary skill in the art will readily be able to determine appropriate doses and schedules of administration of the subject oligonucleotides, whether the oligonucleotides are to be administered to cells or to subjects.

Administration of sd-rxRNAs, such as trhough intradermal injection or subcutaneous delivery, can be optimized through testing of dosing regimens. In some embodiments, a single administration is sufficient. To further prolong the effect of the administered sd-rxRNA, the sd-rxRNA can be administered in a slow-release formulation or device, as would be familiar to one of ordinary skill in the art. The hydrophobic nature of sd-rxRNA compounds can enable use of a wide variety of polymers, some of which are not compatible with conventional oligonucleotide delivery.

In other embodiments, the sd-rxRNA is administered multiple times. In some instances it is administered daily, bi-weekly, weekly, every two weeks, every three weeks, monthly, every two months, every three months, every four months, every five months, every six months or less frequently than every six months. In some instances, it is administered multiple times per day, week, month and/or year. For example, it can be administered approximately every hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours. 7 hours, 8 hours, 9 hours 10 hours, 12 hours or more than twelve hours. It can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 times per day.

Aspects of the invention relate to administering sd-rxRNA molecules to a subject. In some instances the subject is a patient and administering the sd-rxRNA molecule involves administering the sd-rxRNA molecule in a doctor's office.

In some embodiments, more than one sd-rxRNA molecule is administered simultaneously. For example a composition may be administered that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 different sd-rxRNA molecules. In certain embodiments, a composition comprises 2 or 3 different sd-rxRNA molecules. When a composition comprises more than one sd-rxRNA, the sd-rxRNA molecules within the composition can be directed to the same gene or to different genes.

FIG. 1 reveals the expression profile for several genes associated with the invention. As expected, target gene expression is elevated early and returns to normal by day 10. FIG. 2 provides a summary of experimental design. FIGS. 3-6 show in vivo silencing of MAP4K4 and PPIB expression following intradermal injection of sd-rxRNA molecules targeting these genes. FIGS. 7-8 show that the silencing effect of sd-rxRNAs can persist for at least 8 days. Thus, in some embodiments, sd-rxRNA is administered within 8 days prior to an event that compromises or damages the skin such as a surgery. For examples, an sd-rxRNA could be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or more than 10 days prior to an event that compromises or damages the skin. FIG. 9 demonstrates examples of dosing regimens.

In some instances, the effective amount of sd-rxRNA that is delivered by subcutaneous administration is at least approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more than 100 mg/kg including any intermediate values.

In some instances, the effective amount of sd-rxRNA that is delivered through intradermal injection is at least approximately 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or more than 950 pg including any intermediate values.

sd-rxRNA molecules administered through methods described herein are effectively targeted to all the cell types in the skin.

Physical methods of introducing nucleic acids include injection of a solution containing the nucleic acid, bombardment by particles covered by the nucleic acid, soaking the cell or organism in a solution of the nucleic acid, or electroporation of cell membranes in the presence of the nucleic acid. A viral construct packaged into a viral particle would accomplish both efficient introduction of an expression construct into the cell and transcription of nucleic acid encoded by the expression construct. Other methods known in the art for introducing nucleic acids to cells may be used, such as lipid-mediated carrier transport, chemical-mediated transport, such as calcium phosphate, and the like. Thus the nucleic acid may be introduced along with components that perform one or more of the following activities: enhance nucleic acid uptake by the cell, inhibit annealing of single strands, stabilize the single strands, or other-wise increase inhibition of the target gene.

Nucleic acid may be directly introduced into the cell (i.e., intracellularly); or introduced extracellularly into a cavity, interstitial space, into the circulation of an organism, introduced orally, or may be introduced by bathing a cell or organism in a solution containing the nucleic acid. Vascular or extravascular circulation, the blood or lymph system, and the cerebrospinal fluid are sites where the nucleic acid may be introduced.

The cell with the target gene may be derived from or contained in any organism. The organism may a plant, animal, protozoan, bacterium, virus, or fungus. The plant may be a monocot, dicot or gymnosperm; the animal may be a vertebrate or invertebrate. Preferred microbes are those used in agriculture or by industry, and those that are pathogenic for plants or animals.

Alternatively, vectors, e.g., transgenes encoding a siRNA of the invention can be engineered into a host cell or transgenic animal using art recognized techniques.

A further preferred use for the agents of the present invention (or vectors or transgenes encoding same) is a functional analysis to be carried out in eukaryotic cells, or eukaryotic non-human organisms, preferably mammalian cells or organisms and most preferably human cells, e.g. cell lines such as HeLa or 293 or rodents, e.g. rats and mice. By administering a suitable priming agent/RNAi agent which is sufficiently complementary to a target mRNA sequence to direct target-specific RNA interference, a specific knockout or knockdown phenotype can be obtained in a target cell. e.g. in cell culture or in a target organism.

Thus, a further subject matter of the invention is a eukaryotic cell or a eukaryotic non-human organism exhibiting a target gene-specific knockout or knockdown phenotype comprising a fully or at least partially deficient expression of at least one endogenous target gene wherein said cell or organism is transfected with at least one vector comprising DNA encoding an RNAi agent capable of inhibiting the expression of the target gene. It should be noted that the present invention allows a target-specific knockout or knockdown of several different endogenous genes due to the specificity of the RNAi agent.

Gene-specific knockout or knockdown phenotypes of cells or non-human organisms, particularly of human cells or non-human mammals may be used in analytic to procedures, e.g. in the functional and/or phenotypical analysis of complex physiological processes such as analysis of gene expression profiles and/or proteomes. Preferably the analysis is carried out by high throughput methods using oligonucleotide based chips.

Assays of Oligonucleotide Stability

In some embodiments, the oligonucleotides of the invention are stabilized, i.e., substantially resistant to endonuclease and exonuclease degradation. An oligonucleotide is defined as being substantially resistant to nucleases when it is at least about 3-fold more resistant to attack by an endogenous cellular nuclease, and is highly nuclease resistant when it is at least about 6-fold more resistant than a corresponding oligonucleotide. This can be demonstrated by showing that the oligonucleotides of the invention are substantially resistant to nucleases using techniques which are known in the art.

One way in which substantial stability can be demonstrated is by showing that the oligonucleotides of the invention function when delivered to a cell, e.g., that they reduce transcription or translation of target nucleic acid molecules, e.g., by measuring protein levels or by measuring cleavage of mRNA. Assays which measure the stability of target RNA can be performed at about 24 hours post-transfection (e.g., using Northern blot techniques, RNase Protection Assays, or QC-PCR assays as known in the art). Alternatively, levels of the target protein can be measured. Preferably, in addition to testing the RNA or protein levels of interest, the RNA or protein levels of a control, non-targeted gene will be measured (e.g., actin, or preferably a control with sequence similarity to the target) as a specificity control. RNA or protein measurements can be made using any art-recognized technique. Preferably, measurements will be made beginning at about 16-24 hours post transfection. (M. Y. Chiang, et al. 1991. J Biol Chem. 266:18162-71; T. Fisher, et al. 1993. Nucleic Acids Research. 21 3857).

The ability of an oligonucleotide composition of the invention to inhibit protein synthesis can be measured using techniques which are known in the art, for example, by detecting an inhibition in gene transcription or protein synthesis. For example. Nuclease SI mapping can be performed. In another example, Northern blot analysis can be used to measure the presence of RNA encoding a particular protein. For example, total RNA can be prepared over a cesium chloride cushion (see, e.g., Ausebel et al., 1987. Current Protocols in Molecular Biology (Greene & Wiley, New York)). Northern blots can then be made using the RNA and probed (see, e.g., Id.). In another example, the level of the specific mRNA produced by the target protein can be measured, e.g., using PCR. In yet another example, Western blots can be used to measure the amount of target protein present. In still another embodiment, a phenotype influenced by the amount of the protein can be detected. Techniques for performing Western blots are well known in the art, see, e.g., Chen et al. J. Biol. Chem. 271:28259.

In another example, the promoter sequence of a target gene can be linked to a reporter gene and reporter gene transcription (e.g., as described in more detail below) can be monitored. Alternatively, oligonucleotide compositions that do not target a promoter can be identified by fusing a portion of the target nucleic acid molecule with a reporter gene so that the reporter gene is transcribed. By monitoring a change in the expression of the reporter gene in the presence of the oligonucleotide composition, it is possible to determine the effectiveness of the oligonucleotide composition in inhibiting the expression of the reporter gene. For example, in one embodiment, an effective oligonucleotide composition will reduce the expression of the reporter gene.

A “reporter gene” is a nucleic acid that expresses a detectable gene product, which may be RNA or protein. Detection of mRNA expression may be accomplished by Northern blotting and detection of protein may be accomplished by staining with antibodies specific to the protein. Preferred reporter genes produce a readily detectable product. A reporter gene may be operably linked with a regulatory DNA sequence such that detection of the reporter gene product provides a measure of the transcriptional activity of the regulatory sequence. In preferred embodiments, the gene product of the reporter gene is detected by an intrinsic activity associated with that product. For instance, the reporter gene may encode a gene product that, by enzymatic activity, gives rise to a detectable signal based on color, fluorescence, or luminescence. Examples of reporter genes include, but are not limited to, those coding for chloramphenicol acetyl transferase (CAT), luciferase, beta-galactosidase, and alkaline phosphatase.

One skilled in the art would readily recognize numerous reporter genes suitable for use in the present invention. These include, but are not limited to, chloramphenicol acetyltransferase (CAT), luciferase, human growth hormone (hGH), and beta-galactosidase. Examples of such reporter genes can be found in F. A. Ausubel et al., Eds., Current Protocols in Molecular Biology, John Wiley & Sons, New York, (1989). Any gene that encodes a detectable product, e.g., any product having detectable enzymatic activity or against which a specific antibody can be raised, can be used as a reporter gene in the present methods.

One reporter gene system is the firefly luciferase reporter system. (Gould, S. J., and Subramani, S. 1988. Anal. Biochem., 7:404-408 incorporated herein by reference). The luciferase assay is fast and sensitive. In this assay, a lysate of the test cell is prepared and combined with ATP and the substrate luciferin. The encoded enzyme luciferase catalyzes a rapid. ATP dependent oxidation of the substrate to generate a light-emitting product. The total light output is measured and is proportional to the amount of luciferase present over a wide range of enzyme concentrations.

CAT is another frequently used reporter gene system; a major advantage of this system is that it has been an extensively validated and is widely accepted as a measure of promoter activity. (Gorman C. M., Moffat, L. F., and Howard, B. H. 1982. Mol. Cell. Biol., 2:1044-1051). In this system, test cells are transfected with CAT expression vectors and incubated with the candidate substance within 2-3 days of the initial transfection. Thereafter, cell extracts are prepared. The extracts are incubated with acetyl CoA and radioactive chloramphenicol. Following the incubation, acetylated chloramphenicol is separated from nonacetylated form by thin layer chromatography. In this assay, the degree of acetylation reflects the CAT gene activity with the particular promoter.

Another suitable reporter gene system is based on immunologic detection of hGH. This system is also quick and easy to use. (Selden. R., Burke-Howie, K. Rowe, M. E., Goodman, H. M., and Moore, D. D. (1986), Mol. Cell, Biol., 6:3173-3179 incorporated herein by reference). The hGH system is advantageous in that the expressed hGH polypeptide is assayed in the media, rather than in a cell extract. Thus, this system does not require the destruction of the test cells. It will be appreciated that the principle of this reporter gene system is not limited to hGH but rather adapted for use with any polypeptide for which an antibody of acceptable specificity is available or can be prepared.

In one embodiment, nuclease stability of a double-stranded oligonucleotide of the invention is measured and compared to a control, e.g., an RNAi molecule typically used in the art (e.g., a duplex oligonucleotide of less than 25 nucleotides in length and comprising 2 nucleotide base overhangs) or an unmodified RNA duplex with blunt ends.

The target RNA cleavage reaction achieved using the siRNAs of the invention is highly sequence specific. Sequence identity may determined by sequence comparison and alignment algorithms known in the art. To determine the percent identity of two nucleic acid sequences (or of two amino acid sequences), the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first sequence or second sequence for optimal alignment). A preferred, non-limiting example of a local alignment algorithm utilized for the comparison of sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into the BLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. Greater than 90% sequence identity, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity, between the siRNA and the portion of the target gene is preferred. Alternatively, the siRNA may be defined functionally as a nucleotide sequence (or oligonucleotide sequence) that is capable of hybridizing with a portion of the target gene transcript. Examples of stringency conditions for polynucleotide hybridization are provided in Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11, and Current Protocols in Molecular Biology, 1995, F. M. Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporated herein by reference.

Therapeutic Use

By inhibiting the expression of a gene, the oligonucleotide compositions of the present invention can be used to treat any disease involving the expression of a protein. Examples of diseases that can be treated by oligonucleotide compositions, just to illustrate, include: cancer, retinopathies, autoimmune diseases, inflammatory diseases (i.e., ICAM-1 related disorders, Psoriasis, Ulcerative Colitus, Crohn's disease), viral diseases (i.e., HIV, Hepatitis C), miRNA disorders, and cardiovascular diseases.

In one embodiment, in vitro treatment of cells with oligonucleotides can be used for ex vivo therapy of cells removed from a subject (e.g., for treatment of leukemia or viral infection) or for treatment of cells which did not originate in the subject, but are to be administered to the subject (e.g., to eliminate transplantation antigen expression on cells to be transplanted into a subject). In addition, in vitro treatment of cells can be used in non-therapeutic settings, e.g., to evaluate gene function, to study gene regulation and protein synthesis or to evaluate improvements made to oligonucleotides designed to modulate gene expression or protein synthesis. In vivo treatment of cells can be useful in certain clinical settings where it is desirable to inhibit the expression of a protein. There are numerous medical conditions for which antisense therapy is reported to be suitable (see, e.g., U.S. Pat. No. 5,830,653) as well as respiratory syncytial virus infection (WO 95/22,553) influenza virus (WO 94/23,028), and malignancies (WO 94/08,003). Other examples of clinical uses of antisense sequences are reviewed, e.g., in Glaser. 1996. Genetic Engineering News 16:1. Exemplary targets for cleavage by oligonucleotides include, e.g., protein kinase Ca, ICAM-1, c-raf kinase, p53, c-myb, and the bcr/abl fusion gene found in chronic myelogenous leukemia.

The subject nucleic acids can be used in RNAi-based therapy in any animal having RNAi pathway, such as human, non-human primate, non-human mammal, non-human vertebrates, rodents (mice, rats, hamsters, rabbits, etc.), domestic livestock animals, pets (cats, dogs, etc.), Xenopus, fish, insects (Drosophila, etc.), and worms (C. elegans), etc.

The invention provides methods for preventing in a subject, a disease or condition associated with an aberrant or unwanted target gene expression or activity, by administering to the subject a therapeutic agent (e.g., a RNAi agent or vector or transgene encoding same). If appropriate, subjects are first treated with a priming agent so as to be more responsive to the subsequent RNAi therapy. Subjects at risk for a disease which is caused or contributed to by aberrant or unwanted target gene expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the target gene aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of target gene aberrancy, for example, a target gene, target gene agonist or target gene antagonist agent can be used for treating the subject.

In another aspect, the invention pertains to methods of modulating target gene expression, protein expression or activity for therapeutic purposes. Accordingly, in an exemplary embodiment, the modulatory method of the invention involves contacting a cell capable of expressing target gene with a therapeutic agent of the invention that is specific for the target gene or protein (e.g., is specific for the mRNA encoded by said gene or specifying the amino acid sequence of said protein) such that expression or one or more of the activities of target protein is modulated. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent), in vivo (e.g., by administering the agent to a subject), or ex vivo. Typically, subjects are first treated with a priming agent so as to be more responsive to the subsequent RNAi therapy. As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant or unwanted expression or activity of a target gene polypeptide or nucleic acid molecule. Inhibition of target gene activity is desirable in situations in which target gene is abnormally unregulated and/or in which decreased target gene activity is likely to have a beneficial effect.

The therapeutic agents of the invention can be administered to individuals to treat (prophylactically or therapeutically) disorders associated with aberrant or unwanted target gene activity. In conjunction with such treatment, pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in determining whether to administer a therapeutic agent as well as tailoring the dosage and/or therapeutic regimen of treatment with a therapeutic agent. Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, for example. Eichelbaum. M. et al. (1996) Clin. Exp. Pharmacol. Physiol. 23(10-11): 983-985 and Linder, M. W. et al. (1997) Clin. Chem. 43(2):254-266

RNAi in Skin Indications

Nucleic acid molecules, or compositions comprising nucleic acid molecules, described herein may in some embodiments be administered to pre-treat, treat or prevent compromised skin. As used herein “compromised skin” refers to skin which exhibits characteristics distinct from normal skin. Compromised skin may occur in association with a dermatological condition. Several non-limiting examples of dermatological conditions include rosacea, common acne, seborrheic dermatitis, perioral dermatitis, acneform rashes, transient acantholytic dermatosis, and acne necrotica miliaris. In some instances, compromised skin may comprise a wound and/or scar tissue. In some instances, methods and compositions associated with the invention may be used to promote wound healing, prevention, reduction or inhibition of scarring, and/or promotion of re-epithelialisation of wounds.

A subject can be pre-treated or treated prophylactically with a molecule associated with the invention, prior to the skin of the subject becoming compromised. As used herein “pre-treatment” or “prophylactic treatment” refers to administering a nucleic acid to the skin prior to the skin becoming compromised. For example, a subject could be pre-treated 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 24 hours, 48 hours, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days or more than 8 days prior to the skin becoming compromised. In other embodiments, a subject can be treated with a molecule associated with the invention immediately before the skin becomes compromised and/or simultaneous to the skin becoming compromised and/or after the skin has been compromised. In some embodiments, the skin is compromised through a medical procedure such as surgery, including elective surgery. In certain embodiments methods and compositions may be applied to areas of the skin that are believed to be at risk of becoming compromised. It should be appreciated that one of ordinary skill in the art would be able to optimize timing of administration using no more than routine experimentation.

In some aspects, methods associated with the invention can be applied to promote healing of compromised skin. Administration can occur at any time up until the compromised skin has healed, even if the compromised skin has already partially healed. The timing of administration can depend on several factors including the nature of the compromised skin, the degree of damage within the compromised skin, and the size of the compromised area. In some embodiments administration may occur immediately after the skin is compromised, or 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 48 hours, or more than 48 hours after the skin has been compromised. Methods and compositions of the invention may be administered one or more times as necessary. For example, in some embodiments, compositions may be administered daily or twice daily. In some instances, compositions may be administered both before and after formation of compromised skin.

Compositions associated with the invention may be administered by any suitable route. In some embodiments, administration occurs locally at an area of compromised skin. For example, compositions may be administered by intradermal injection. Compositions for intradermal injection may include injectable solutions. Intradermal injection may in some embodiments occur around the are of compromised skin or at a site where the skin is likely to become compromised. In some embodiments, compositions may also be administered in a topical form, such as in a cream or ointment. In some embodiments, administration of compositions described herein comprises part of an initial treatment or pre-treatment of compromised skin, while in other embodiments, administration of such compositions comprises follow-up care for an area of compromised skin.

The appropriate amount of a composition or medicament to be applied can depend on many different factors and can be determined by one of ordinary skill in the art through routine experimentation. Several non-limiting factors that might be considered include biological activity and bioavailability of the agent, nature of the agent, mode of administration, half-life, and characteristics of the subject to be treated.

In some aspects, nucleic acid molecules associated with the invention may also be used in treatment and/or prevention of fibrotic disorders, including pulmonary fibrosis, liver cirrhosis, scleroderma and glomerulonephritis, lung fibrosis, liver fibrosis, skin fibrosis, muscle fibrosis, radiation fibrosis, kidney fibrosis, proliferative vitreoretinopathy, restenosis, and uterine fibrosis.

A therapeutically effective amount of a nucleic acid molecule described herein may in some embodiments be an amount sufficient to prevent the formation of compromised skin and/or improve the condition of compromised skin and/or to treat or prevent a fibrotic disorder. In some embodiments, improvement of the condition of compromised skin may correspond to promotion of wound healing and/or inhibition of scarring and/or promotion of epithelial regeneration. The extent of prevention of formation of compromised skin and/or improvement to the condition of compromised skin may in some instances be determined by, for example, a doctor or clinician.

The ability of nucleic acid molecules associated with the invention to prevent the formation of compromised skin and/or improve the condition of compromised skin may in some instances be measured with reference to properties exhibited by the skin. In some instances, these properties may include rate of epithelialisation and/or decreased size of an area of compromised skin compared to control skin at comparable time points.

As used herein, prevention of formation of compromised skin, for example prior to a surgical procedure, and/or improvement of the condition of compromised skin, for example after a surgical procedure, can encompass any increase in the rate of healing in the compromised skin as compared with the rate of healing occurring in a control sample. In some instances, the condition of compromised skin may be assessed with respect to either comparison of the rate of re-epithelialisation achieved in treated and control skin, or comparison of the relative areas of treated and control areas of compromised skin at comparable time points. In some aspects, a molecule that prevents formation of compromised skin or promotes healing of compromised skin may be a molecule that, upon administration, causes the area of compromised skin to exhibit an increased rate of re-epithelialisation and/or a reduction of the size of compromised skin compared to a control at comparable time points. In some embodiments, the healing of compromised skin may give rise to a rate of healing that is 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% greater than the rate occurring in controls.

In some aspects, subjects to be treated by methods and compositions associated with the invention may be subjects who will undergo, are undergoing or have undergone a medical procedure such as a surgery. In some embodiments, the subject may be prone to defective, delayed or otherwise impaired re-epithelialisation, such as dermal wounds in the aged. Other non-limiting examples of conditions or disorders in which wound healing is associated with delayed or otherwise impaired re-epithelialisation include patients suffering from diabetes, patients with polypharmacy, post-menopausal women, patients susceptible to pressure injuries, patients with venous disease, clinically obese patients, patients receiving chemotherapy, patients receiving radiotherapy, patients receiving steroid treatment, and immuno-compromised patients. In some instances, defective re-epithelialisation response can contributes to infections at the wound site, and to the formation of chronic wounds such as ulcers.

In some embodiments, methods associated with the invention may promote the re-epithelialisation of compromised skin in chronic wounds, such as ulcers, and may also inhibit scarring associated with wound healing. In other embodiments, methods associated with the invention are applied to prevention or treatment of compromised skin in acute wounds in patients predisposed to impaired wound healing developing into chronic wounds. In other aspects, methods associated with the invention are applied to promote accelerated healing of compromised skin while preventing, reducing or inhibiting scarring for use in general clinical contexts. In some aspects, this can involve the treatment of surgical incisions and application of such methods may result in the prevention, reduction or inhibition of scarring that may otherwise occur on such healing. Such treatment may result in the scars being less noticeable and exhibiting regeneration of a more normal skin structure. In other embodiments, the compromised skin that is treated is not compromised skin that is caused by a surgical incision. The compromised skin may be subject to continued care and continued application of medicaments to encourage re-epithelialisation and healing.

In some aspects, methods associated with the invention may also be used in the treatment of compromised skin associated with grafting procedures. This can involve treatment at a graft donor site and/or at a graft recipient site. Grafts can in some embodiments involve skin, artificial skin, or skin substitutes. Methods associated with the invention can also be used for promoting epithelial regeneration. As used herein, promotion of epithelial regeneration encompasses any increase in the rate of epithelial regeneration as compared to the regeneration occurring in a control-treated or untreated epithelium. The rate of epithelial regeneration attained can in some instances be compared with that taking place in control-treated or untreated epithelia using any suitable model of epithelial regeneration known in the art. Promotion of epithelial regeneration may be of use to induce effective re-epithelialisation in contexts in which the re-epithelialisation response is impaired, inhibited, retarded or otherwise defective. Promotion of epithelial regeneration may be also effected to accelerate the rate of defective or normal epithelial regeneration responses in patients suffering from epithelial damage.

Some instances where re-epithelialisation response may be defective include conditions such as pemphigus, Hailey-Hailey disease (familial benign pemphigus), toxic epidermal necrolysis (TEN)/Lyell's syndrome, epidermolysis bullosa, cutaneous leishmaniasis and actinic keratosis. Defective re-epithelialisation of the lungs may be associated with idiopathic pulmonary fibrosis (IPF) or interstitial lung disease. Defective re-epithelialisation of the eye may be associated with conditions such as partial limbal stem cell deficiency or corneal erosions. Defective re-epithelialisation of the gastrointestinal tract or colon may be associated with conditions such as chronic anal fissures (fissure in ano), ulcerative colitis or Crohn's disease, and other inflammatory bowel disorders.

In some aspects, methods associated with the invention are used to prevent, reduce or otherwise inhibit compromised skin associated with scarring. This can be applied to any site within the body and any tissue or organ, including the skin, eye, nerves, tendons, ligaments, muscle, and oral cavity (including the lips and palate), as well as internal organs (such as the liver, heart, brain, abdominal cavity, pelvic cavity, thoracic cavity, guts and reproductive tissue). In the skin, treatment may change the morphology and organization of collagen fibers and may result in making the scars less visible and blend in with the surrounding skin. As used herein, prevention, reduction or inhibition of scarring encompasses any degree of prevention, reduction or inhibition in scarring as compared to the level of scarring occurring in a control-treated or untreated wound.

Prevention, reduction or inhibition of compromised skin, such as compromised skin associated with dermal scarring, can be assessed and/or measured with reference to microscopic and/or macroscopic characteristics. Macroscopic characteristics may include color, height, surface texture and stiffness of the skin. In some instances, prevention, reduction or inhibition of compromised skin may be demonstrated when the color, height, surface texture and stiffness of the skin resembles that of normal skin more closely after treatment than does a control that is untreated. Microscopic assessment of compromised skin may involve examining characteristics such as thickness and/or orientation and/or composition of the extracellular matrix (ECM) fibers, and cellularity of the compromised skin. In some instances, prevention, reduction or inhibition of compromised skin may be demonstrated when the thickness and/or orientation and/or composition of the extracellular matrix (ECM) fibers, and/or cellularity of the compromised skin resembles that of normal skin more closely after treatment than does a control that is untreated.

In some aspects, methods associated with the invention are used for cosmetic purposes, at least in part to contribute to improving the cosmetic appearance of compromised skin. In some embodiments, methods associated with the invention may be used to prevent, reduce or inhibit compromised skin such as scarring of wounds covering joints of the body. In other embodiments, methods associated with the invention may be used to promote accelerated wound healing and/or prevent, reduce or inhibit scarring of wounds at increased risk of forming a contractile scar, and/or of wounds located at sites of high skin tension.

In some embodiments, methods associated with the invention can be applied to promoting healing of compromised skin in instances where there is an increased risk of pathological scar formation, such as hypertrophic scars and keloids, which may have more pronounced deleterious effects than normal scarring. In some embodiments, methods described herein for promoting accelerated healing of compromised skin and/or preventing, reducing or inhibiting scarring are applied to compromised skin produced by surgical revision of pathological scars.

Aspects of the invention can be applied to compromised skin caused by burn injuries. Healing in response to burn injuries can lead to adverse scarring, including the formation of hypertrophic scars. Methods associated with the invention can be applied to treatment of all injuries involving damage to an epithelial layer, such as injuries to the skin in which the epidermis is damaged. Other non-limiting examples of injuries to epithelial tissue include injuries involving the respiratory epithelia, digestive epithelia or epithelia surrounding internal tissues or organs.

RNAi to Treat Liver Fibrosis

In some embodiments, methods associated with the invention are used to treat liver fibrosis. Liver fibrosis is the excessive accumulation of extracellular matrix proteins, including collagen, that occurs in most types of chronic liver diseases. It is the scarring process that represents the liver's response to injury. Advanced liver fibrosis results in cirrhosis, liver failure, and portal hypertension and often requires liver transplantation. In the same way as skin and other organs heal wounds through deposition of collagen and other matrix constituents so the liver repairs injury through the deposition of new collagen. Activated hepatic stellate cells, portal fibroblasts, and myofibroblasts of bone marrow origin have been identified as major collagen-producing cells in the injured liver. These cells are activated by fibrogenic cytokines such as TGF-β1, angiotensin II, and leptin. In some embodiments, methods provided herein are aimed at inhibiting the accumulation of fibrogenic cells and/or preventing the deposition of extracellular matrix proteins. In some embodiments, RNAi molecules (including sd-rxRNA and rxRNAori) may be designed to target CTGF, TGF-β1, angiotensin II, and/or leptin. In some embodiments. RNAi molecules (including sd-rxRNA and rxRNAori) may be designed to target those genes listed in Tables 1-25.

Trabeculectomy Failure

Trabeculectomy is a surgical procedure designed to create a channel or bleb though the sclera to allow excess fluid to drain from the anterior of the eye, leading to reduced intracocular pressure (IOP), a risk factor for glaucoma-related vision loss. The most common cause of trabeculectomy failure is blockage of the bleb by scar tissue. In certain embodiments, the sd-rxRNA is used to prevent formation of scar tissue resulting from a trabeculectomy. In some embodiments, the sd-rxRNA targets connexin 43. In other embodiments, the sd-rxRNA targets proyly 4-hydroxylase. In yet other embodiments, the sd-rxRNA targets procollagen C-protease.

Target Genes

It should be appreciated that based on the RNAi molecules designed and disclosed herein, one of ordinary skill in the art would be able to design such RNAi molecules to target a variety of different genes depending on the context and intended use. For purposes of pre-treating, treating, or preventing compromised skin and/or promoting wound healing and/or preventing, reducing or inhibiting scarring, one of ordinary skill in the art would appreciate that a variety of suitable target genes could be identified based at least in part on the known or predicted functions of the genes, and/or the known or predicted expression patterns of the genes. Several non-limiting examples of genes that could be targeted by RNAi molecules for pre-treating, treating, or preventing compromised skin and/or promoting wound healing and/or preventing, Ill reducing or inhibiting scarring include genes that encode for the following proteins: Transforming growth factor β (TGFβ1, TGFβ2, TGFβ3), Osteopontin (SPP1), Connective tissue growth factor (CTGF), Platelet-derived growth factor (PDGF), Hypoxia inducible factor-1α (HIF1α), Collagen I and/or III, Prolyl 4-hydroxylase (P4H). Procollagen C-protease (PCP), Matrix metalloproteinase 2, 9 (MMP2, 9), Integrins, Connexin, Histamine H1 receptor, Tissue transglutaminase, Mammalian target of rapamycin (mTOR), HoxB13, VEGF, IL-6. SMAD proteins, Ribosomal protein S6 kinases (RSP6), Cyclooxygenase-2 (COX-2/PTGS2), Cannabinoid receptors (CB1, CB2), and/or miR29b.

Transforming growth factor β proteins, for which three isoforms exist in mammals (TGFβ1, TGFβ2, TGFβ3), are secreted proteins belonging to a superfamily of growth factors involved in the regulation of many cellular processes including proliferation, migration, apoptosis, adhesion, differentiation, inflammation, immuno-suppression and expression of extracellular proteins. These proteins are produced by a wide range of cell types including epithelial, endothelial, hematopoietic, neuronal, and connective tissue cells. Representative Genbank accession numbers providing DNA and protein sequence information for human TGFβ1, TGFβ2 and TGFβ3 are BT007245, BC096235, and X14149, respectively. Within the TGFβ family, TGFβ1 and TGFβ2 but not TGFβ3 represent suitable targets. The alteration in the ratio of TGFβ variants will promote better wound healing and will prevent excessive scar formation.

Osteopontin (OPN), also known as Secreted phosphoprotein 1 (SPP1), Bone Sinaloprotein 1 (BSP-1), and early T-lymphocyte activation (ETA-1) is a secreted glycoprotein protein that binds to hydroxyapatite. OPN has been implicated in a variety of biological processes including bone remodeling, immune functions, chemotaxis, cell activation and apoptosis. Osteopontin is produced by a variety of cell types including fibroblasts, preosteoblasts, osteoblasts, osteocytes, odontoblasts, bone marrow cells, hypertrophic chondrocytes, dendritic cells, macrophages, smooth muscle, skeletal muscle myoblasts, endothelial cells, and extraosseous (non-bone) cells in the inner ear, brain, kidney, deciduum, and placenta. Representative Genbank accession number providing DNA and protein sequence information for human Osteopontin are NM_000582.2 and X13694.

Connective tissue growth factor (CTGF), also known as Hypertrophic chondrocyte-specific protein 24, is a secreted heparin-binding protein that has been implicated in wound healing and scleroderma. Connective tissue growth factor is active in many cell types including fibroblasts, myofibroblasts, endothelial and epithelial cells. Representative Genbank accession number providing DNA and protein sequence information for human CTGF are NM_001901.2 and M92934.

The Platelet-derived growth factor (PDGF) family of proteins, including several isoforms, are secreted mitogens. PDGF proteins are implicated in wound healing, at least in part, because they are released from platelets following wounding. Representative Genbank accession numbers providing DNA and protein sequence information for human PDGF genes and proteins include X03795 (PDGFA). X02811 (PDGFB), AF091434 (PDGFC), AB033832 (PDGFD).

Hypoxia inducible factor-1α (HIF1α), is a transcription factor involved in cellular response to hypoxia. HIF1α is implicated in cellular processes such as embryonic vascularization, tumor angiogenesis and pathophysiology of ischemic disease. A representative Genbank accession number providing DNA and protein sequence information for human HIF1α is U22431.

Collagen proteins are the most abundant mammalian proteins and are found in tissues such as skin, tendon, vascular, ligature, organs, and bone. Collagen I proteins (such as COL1A1 and COL1A2) are detected in scar tissue during wound healing, and are expressed in the skin. Collagen III proteins (including COL3A 1) are detected in connective tissue in wounds (granulation tissue), and are also expressed in skin. Representative Genbank accession numbers providing DNA and protein sequence information for human Collagen proteins include: Z74615 (COL1A1), J03464 (COL1A2) and X14420 (COL3A1).

Prolyl 4-hydroxylase (P4H), is involved in production of collagen and in oxygen sensing. A representative Genbank accession number providing DNA and protein sequence information for human P4H is AY198406.

Procollagen C-protease (PCP) is another target.

Matrix metalloproteinase 2, 9 (MMP2, 9) belong to the metzincin metalloproteinase superfamily and are zinc-dependent endopeptidases. These proteins are implicated in a variety of cellular processes including tissue repair. Representative Genbank accession numbers providing DNA and protein sequence information for human MMP proteins are M55593 (MMP2) and J05070 (MMP9).

Integrins are a family of proteins involved in interaction and communication between a cell and the extracellular matrix. Vertebrates contain a variety of integrins including α₁β₁, α₂β₁, α₄β₁, α₅β₁, α₆β₁, α_(L)β₂, α_(M)β₂, α_(IIb)β₃, α_(v)β₃, α_(v)β₅, α₆β₄.

Connexins are a family of vertebrate transmembrane proteins that form gap junctions. Several examples of Connexins, with the accompanying gene name shown in brackets, include Cx23 (GJE1), Cx25 (GJB7), Cx26 (GJB2), Cx29 (GJE1), Cx30 (GJB6), Cx30.2 (GJC3), Cx30.3 (GJB4), Cx31 (GJB3), Cx31.1 (GJB5), Cx31.9 (GJC1/GJD3), Cx32 (GJB1), Cx33 (GJA6), Cx36 (GJD2/GJA9), Cx37 (GJA4), Cx39 (GJD4), Cx40 (GJA5), Cx40.1 (GJD4), Cx43 (GJA1), Cx45 (GJC1/GJA7), Cx46 (GJA3), Cx47 (GJC2/GJA12), Cx50 (GJA8), Cx59 (GJA10), and Cx62 (GJA10).

Histamine H1 receptor (HRH1) is a metabotropic G-protein-coupled receptor involved in the phospholipase C and phosphatidylinositol (PIP2) signaling pathways. A representative Genbank accession number providing DNA and protein sequence information for human HRH1 is Z34897.

Tissue transglutaminase, also called Protein-glutamine gamma-glutamyltransferase 2, is involved in protein crosslinking and is implicated is biological processes such as apoptosis, cellular differentiation and matrix stabilization. A representative Genbank accession number providing DNA and protein sequence information for human Tissue transglutaminase is M55153.

Mammalian target of rapamycin (mTOR), also known as Serine/threonine-protein kinase mTOR and FK506 binding protein 12-rapamycin associated protein 1 (FRAPI), is involved in regulating cell growth and survival, cell motility, transcription and translation. A representative Genbank accession number providing DNA and protein sequence information for human mTOR is L34075.

HoxB13 belongs to the family of Homeobox proteins and has been linked to functions such as cutaneous regeneration and fetal skin development. A representative Genbank accession number providing DNA and protein sequence information for human HoxB13 is U57052.

Vascular endothelial growth factor (VEGF) proteins are growth factors that bind to tyrosine kinase receptors and are implicated in multiple disorders such as cancer, age-related macular degeneration, rheumatoid arthritis and diabetic retinopathy. Members of this protein family include VEGF-A, VEGF-B, VEGF-C and VEGF-D. Representative Genbank accession numbers providing DNA and protein sequence information for human VEGF proteins are M32977 (VEGF-A). U43368 (VEGF-B), X94216 (VEGF-C), and D89630 (VEGF-D).

Interleukin-6 (IL-6) is a cytokine involved in stimulating immune response to tissue damage. A representative Genbank accession number providing DNA and protein sequence information for human IL-6 is X04430.

SMAD proteins (SMAD1-7, 9) are a family of transcription factors involved in regulation of TGFβ signaling. Representative Genbank accession numbers providing DNA and protein sequence information for human SMAD proteins are U59912 (SMAD1), U59911 (SMAD2), U68019 (SMAD3), U44378 (SMAD4), U59913 (SMAD5), U59914 (SMAD6), AF015261 (SMAD7), and BC011559 (SMAD9).

Ribosomal protein S6 kinases (RSK6) represent a family of serine/threonine kinases involved in activation of the transcription factor CREB. A representative Genbank accession number providing DNA and protein sequence information for human Ribosomal protein S6 kinase alpha-6 is AF184965.

Cyclooxygenase-2 (COX-2), also called Prostaglandin G/H synthase 2 (PTGS2), is involved in lipid metabolism and biosynthesis of prostanoids and is implicated in inflammatory disorders such as rheumatoid arthritis. A representative Genbank accession number providing DNA and protein sequence information for human COX-2 is AY462100.

Cannabinoid receptors, of which there are currently two known subtypes, CB1 and CB2, are a class of cell membrane receptors under the G protein-coupled receptor superfamily. The CB1 receptor is expressed mainly in the brain, but is also expressed in the lungs, liver and kidneys, while the CB2 receptor is mainly expressed in the immune system and in hematopoietic cells. A representative Genbank accession number providing DNA and protein sequence information for human CB1 is NM_001160226. NM_001160258, NM_001160259, NM_001160260, NM_016083, and NM_033181.

miR29b (or miR-29b) is a microRNA (miRNA), which is a short (20-24 nt) noncoding RNA involved in post-transcriptional regulation of gene expression in multicellular organisms by affecting both the stability and translation of mRNAs. miRNAs are transcribed by RNA polymerase II as part of capped and polyadenylated primary transcripts (pri-miRNAs) that can be either protein-coding or non-coding. The primary transcript is cleaved by the Drosha ribonuclease III enzyme to produce an approximately 70-nt stem-loop precursor miRNA (pre-miRNA), which is further cleaved by the cytoplasmic Dicer ribonuclease to generate the mature miRNA and antisense miRNA star (miRNA*) products. The mature miRNA is incorporated into a RNA-induced silencing complex (RISC), which recognizes target mRNAs through imperfect base pairing with the miRNA and most commonly results in translational inhibition or destabilization of the target mRNA. A representative miRBase accession number for miR29b is MI0000105 (website: mirbase.org/cgi-bin/mirna_entry.pl?acc=MI0000105).

In some embodiments, the sd-rxRNA targets connexin 43 (CX43). This gene is a member of the connexin gene family. The encoded protein is a component of gap junctions, which are composed of arrays of intercellular channels that provide a route for the diffusion of low molecular weight materials from cell to cell. The encoded protein is the major protein of gap junctions in the heart that are thought to have a crucial role in the synchronized contraction of the heart and in embryonic development. A related intronless pseudogene has been mapped to chromosome 5. Mutations in this gene have been associated with oculodentodigital dysplasia and heart malformations. Representative Genbank accession numbers providing DNA and protein sequence information for human CX43 genes and proteins include NM_000165 and NP_000156.

In other embodiments, the sd-rxRNA targets prolyl 4-hydroxylase (P4HTM). The product of this gene belongs to the family of prolyl 4-hydroxylases. This protein is a prolyl hydroxylase that may be involved in the degradation of hypoxia-inducible transcription factors under normoxia. It plays a role in adaptation to hypoxia and may be related to cellular oxygen sensing. Alternatively spliced variants encoding different isoforms have been identified. Representative Genbank accession numbers providing DNA and protein sequence information for human P4HTM genes and proteins include NM_177938. NP_808807. NM_177939, and NP_808808.

In certain embodiments, the sd-rxRNA targets procollagen C-protease.

EXAMPLES Example 1: In Vivo Gene Silencing in Skin after Local Delivery of Sd-rxRNA

Demonstrated herein is gene silencing in skin following administration of sd-rxRNA molecules. Rat incision models were used which included 6 dorsal incisions per animal. Analysis included monitoring of digital images, detection of target gene expression, scar assessment, and histology. FIG. 1 reveals an expression profile of several genes including MAP4K4, SPP1, CTGF, PTGS2 and TGFB1. As expected, when expression of these genes was monitored post-incision, target gene expression ws elevated early and then returned to normal by day 10.

FIG. 2 presents an overview of intradermal injection experiments wih sd-rxRNA molecules. 6 intradermal injections were performed at each site. Each injection consisted of approximately 34μ, 300 μg total. Images were taken before injection and 15 minutes after the first injection.

FIG. 3 demonstrates in vivo silencing following intradermal injection of sd-rxRNA in rats. 6 injections were made per dose. 300 μg in PBS was injected on days 1 & 2 (2 doses) or on day 2 (1 dose). 5 incisions sites were made per treatment. Incisions were 1 cm. 3 mm skin biopsies were harvested 48 hours after the last dose and target expression was determined by QPCR.

FIG. 4 demonstrates in vivo silencing of MAP4K4, PPIB and CTGF expression in rats following intradermal injection of sd-rxRNA molecules. A single intradermal injection of PBS (vehicle), or 300 ug of MAP4K4, or 2 different CTGF or PPIB targeting sd-rxRNA were injected at 6 sites. 3 mm skin biopsies harvested 48 hours post injection and processed for RNA. Data was analyzed by QPCR and normalized to B-Actin. PBS was set to 1. Data was graphed as a percent reduction in targeted gene expression relative to non-targeting sd-rxRNA (i.e. targeting other gene). Gene expression from untreated skin samples on treated animals are similar to PBS treated or sham controls.

FIG. 5 demonstrates in vivo silencing in mice following intradermal injection of sd-rxRNA molecules. C57BL/6 mice were used, with n=7 wheal sites active and PBS. The control group consisted of 12. 300 ug was administered in 50 ul/injections. 3 mm biopsies were processed for RNA, and target expression determined by QPCR. Expression was normalized to housekeeping gene cyclophilin B.

FIG. 6 reveals the in vitro potency and in vivo effectiveness of 2 different sd-rxRNAs targeting PPIB. Two PPIB sd-rxRNAs with different EC50s were compared in vivo. Similar in vivo results were obtained with 1 injection of 300 μg

FIGS. 7 and 8 demonstrate the duration of gene silencing achieved through administration of sd-rxRNA. There were 6 injection sites per animal. 3 mm skin biopsies were harvested on days 3, 5, and 8. RNA was isolated and gene expression was analyzed by qPCR and normalized to B-Actin

FIG. 9 compares two different dosage regimens, Days 1 and 3 vs. Days 0 and 2. There were 6 injection sites per animal. 3 mm skin biopsies were harvested on days 3, 5, and 8. RNA was isolated and gene expression was analyzed by qPCR and normalized to B-Actin.

Example 2: Identification of Potent Sd-rxRNAs

Up to 300 rxRNA ori compounds were screened for 5 anti-scarring targets. Optimal sequences in SPP1, CTGF, PTGS2, TGFB1 and TGFB2 for sd-rxRNA development were identified using a sequence selection algorithm. The algorithm selects sequences based on the following criteria: a GC content greater than 32% but less than 47%, homology to specific animal models (e.g., mouse or rat), avoidance of 5 or more U/U stretches and/or 2 or more G/C stretches, an off-target hit score of less than 500, and avoidance of sequences contained within the 5′ UTR.

The sequences were developed initially as 25 nucleotide blunt-ended duplexes with O-methyl modification. Such sequences were screened in various cell lines to identify those were most efficient in reducing gene expression. Several concentrations of the RNA molecules, such as 0.025, 0.1 and 0.25 nM, were tested, and suitable concentrations to screen for bDNA were determined. A bDNA was then run of a full screen at a desired concentration. Dose response curves were generated to determine the most potent sequences. Hyperfunctional hits were those with an EC50 of less than 100 pM in lipid transfection. Potent molecules were selected to be developed into sd-rxRNAs based on the parameters described throughout the application and a secondary screen was conducted using the sd-rxRNAs.

FIGS. 10-12 reveal that CTGF sd-rxRNAs are efficacious in mediating gene silencing. A dose response for CTGF is indicated in FIG. 12.

FIGS. 13-14 reveal that the original sd-rxrNA screen had a low hit rate. FIG. reveals PTGS2 knockdown using sd-rxRNA against PTGS2. FIGS. 16-24 reveal that hTGFB1, TGFB, TGFB2 sd-rxRNAs are capable of mediating gene silencing. FIGS. 25-28 shows the identification of potent hSPP1 sd-rxRNAs.

Example 3: Linker Chemistry

FIG. 36 demonstrates that variation of linker chemistry does not influence silencing activity of sd-rxRNAs in vitro. Two different linker chemistries were evaluated, a hydroxyproline linker and ribo linker, on multiple sd-rxRNAs (targeting Map4k4 or PPIB) in passive uptake assays to determine linkers which favor self delivery. HeLa cells were transfected in the absence of a delivery vehicle (passive transfection) with sd-rxRNAs at 1 uM, 0.1 uM or 0.01 uM for 48 hrs. Use of either linker results in an efficacious delivery of sd-rxRNA.

The ribo linker used in Example 5 had the following structure:

Example 4: Optimization of Target Sequences

Chemical optimization was performed for several lead sequences, including CTGF, PTGS2, TGFβ1, and TGFβ2. Multiples versions of sd-rxRNA leads were synthesized. The sense strand was further O-methyl modified, such as by introduction of O-methyl blocks on the ends, introduction of O-methyl phosphorothioate blocks at the ends or introduction of ful O-methyl modification with a phosphorothioate block on the 3′end.

The guide strand was modified to decrease the number of 2′F, substitute 2′F with O-methyl, vary the number of ribonucleotides, eliminate stretches of ribonucleotides, minimize the presence of ribonucleotides next to the phosphorothioate modifications, and if possible remove ribonucleotides from the single stranded region.

Various versions of compounds were synthesized and their efficacy was tested in vitro using passive uptake. The efficacy and toxicity of the optimized compounds was evaluated in vivo.

All compounds show in vivo efficacy. Initially, activity required high concentration and at high concentrations some compound demonstrated injection site reaction. However, data indicated that efficacy and toxicity in vivo could be dramatically improved by enhancement of stability and reduction of 2′ F content. In some instances, toxicity, at least in part, was related to the presence of cholesterol containing short oligomer metabolites. This type of toxicity is expected to be reduced by stabilization. In general, chemical stabilization was well tolerated. Exact chemical optimization patterns differed for various compound. In some cases, complete stabilization resulted in a slightly negative impact on activity. For most target sites, at least two chemically optimized leads were identified: chemically optimized with in vitro efficacy retained or improved compared to an Early Lead and Fully Modified, where in vitro efficacy is slightly reduced.

In general, a fully O-methyl modified sense strand is acceptable. In some instances, it is preferable if less than all of the nucleotides in the sense strand are O-methyl modified. In some instances, the 3′ end of the passenger strand contained a PSI O-METHYL block (2 O-methyl modifications and two 2 phosphorothioate modifications) to insure maximized stability next to the hydrophobic conjugate.

For all compounds, it was possible to identify functional heavily stabilized leads. In some instances, the number of ribonucleotides per compounds was reduced to 4-6. Multiples versions of sd-rxRNA leads were synthesized. The number of 2′F modified purines was limited where possible to improve manufacturability but some optimized compounds do contain some 2′F modified purines.

Optimized Compounds

A summary of CTGF lead compounds is shown in Table 24. PTGS leads are shown in Table 25. hTGFβ1 leads are shown in Table 26 and hTGFβ2 leads are shown in Table 27. Lead compounds were tested for in vitro efficacy with varying levels of methylation of the sense strand.

For CTGF Lead 1 (L1), the fully O-methyl modified sense strand was efficacious having a slight reduction in in vitro efficacy.

For CTGF L2, the fully O-methyl modified sense strand was efficacious, having a slight reduction in in vitro efficacy.

For CTGF L3, the fully O-methyl modified sense strand was partially efficacious, having a reduction in in vitro efficacy.

For CTGF L4, the fully O-methyl modified sense strand was partially efficacious, having a slight reduction in in vitro efficacy.

For PTGS2 L1 and L2, the fully O-methyl modified sense strand was not efficacious.

For TGFβ1 hL3, the fully O-methyl modified sense strand was efficacious.

For TGFβ2, the fully O-methyl modified sense strand was efficacious.

In Vivo Efficacy of Lead Compounds

The activity of lead compounds was tested in vivo both in cell culture and in animal models. FIGS. 33 and 34 demonstrate the activity of optimized CTGF L1 compounds. FIG. 35 demonstrates the in vitro stability of the CTGF L1 compounds. FIGS. 36 and 37 demonstrate the activity of optimized CTGF L2 compounds. FIG. 38 demonstrates the in vitro stability of the CTGF L2 compounds. FIG. 39 provides a summary of the in vivo activity of CTGF lead compounds. FIG. 40 demonstrates the efficacy of CTGF L1 compounds in skin biopsies from rats. FIG. 41 shows the efficacy of CTGF L2 compounds in achieving gene silencing.

FIG. 42 demonstrates CTGF silencing following intradermal injection of RXi-109. FIG. 43 demonstrates the duration of CTGF silencing in skin after intradermal injection of the sd-rxRNA in SD rats. Eight millimeter skin biopsies were harvested, and mRNA levels were quantified by QPCR and normalized to a housekeeping gene. Shown is percent (%) silencing vs. Non Targeting Control (NTC); PBS at each time point is one experimental group; * p≤0.04; ** p≤0.002.

FIGS. 44-46 show that CTGF L3 and LA compounds are also active. FIG. 47 demonstrates changes in mRNA expression levels of CTGF, α-SM actin, collagen 1A2, and collagen 3A1 after intradermal injection of CTFG sd-rxRNA in SD rats. mRNA levels were quantified by qPCR. Substantial reduction in CTGF expression is observed.

FIG. 49 demonstrates that administration of sd-rxRNAs decreases wound width over the course of at least 9 days. The graph shows microscopic measurements of wound width in rats on days 3, 6, and 9 post-wounding. Each group represents 5 rats. Two non-serial sections from each wound were measured and the average width of the two was calculated per wound. *p<0.05 vs. PBS an NTC.

FIG. 50 demonstrates that administration of sd-rxRNAs decreases wound area over the course of at least 9 days. The graph shows microscopic measurements of wound width in rats on days 3, 6, and 9 post-wounding. Each group represents 5 rats. Two non-serial sections from each wound were measured and the average width of the two was calculated per wound. *p<0.05 vs. PBS an NTC.

FIG. 51 demonstrates that administration of sd-rxRNAs increase the percentage of wound re-epithelialization over the course of at least 9 days. The graph shows microscopic measurements of wound width in rats on days 3, 6, and 9 post-wounding. Each group represents 5 rats. Two non-serial sections from each wound were measured and the average width of the two was calculated per wound. *p<0.05 vs. PBS an NTC.

FIG. 52 demonstrates that administration of sd-rxRNAs increases the average granulation tissue maturity scores over the course of at least 9 days. The graph shows microscopic measurements of wound width in rats on days 3, 6, and 9 post-wounding (5=mature. I=immature). Each group represents 5 rats. FIG. 53 demonstrates CD68 labeling in day 9 wounds (0=no labeling, 3=substantial labeling). Each group represents 5 rats.

FIG. 54 demonstrates that CTGF leads have different toxicity levels in vitro. FIG. 55 shows percentage (%) of cell viability after RXi-109 dose escalation (oligos formulated in PBS).

FIG. 56 is a schematic of a non-limiting example of a Phase 1 and 2 clinical trial design for lead compounds. This schematic represents a divided dose, single day ascending dose clinical trial.

FIG. 57 is a schematic of a non-limiting example of a Phase 1 and 2 clinical trial design. This schematic represents a divided dose, multi-day ascending dose clinical trial.

Activity of PTGS2, TGFβ1 and TGFβ2 leads was also tested. FIGS. 59 and 60 demonstrate activity of PTGS2 L1 and L2 compounds. FIGS. 61 and 62 demonstrate the activity of h TGFβ1 compounds and FIGS. 63 and 64 demonstrate the activity of hTGFβ2 compounds.

Gene knock-down in liver was also tested following tail vein injection mice. FIG. 58 demonstrates a percent (%) decrease in PPIB expression in the liver relative to PBS control. Lipoid formulated rxRNAs (10 mg/kg) were delivery systemically to Balb/c mice (n=5) by single tail vein injections. Liver tissue was harvested at 24 hours after injection and expression was analyzed by qPCR (normalized to β-actin). Map4K4 rxRNAori also showed significant silencing (˜83%, p<0.001) although Map4K4 sd-rxRNA did not significantly reduce target gene expression (˜17%, p=0.019). TD.035.2278, Published lipidoid delivery reagent, 98N12-5(1), from Akinc, 2009.

Table 1 provides sequences tested in the Original sd-rxRNA screen.

Table 2 demonstrates inhibition of gene expression with PTGS2 ori sequences.

Table 3 demonstrates non-limiting examples of PTGS2 sd-rxRNA sequences.

Table 4 demonstrates non-limiting examples of TGFB1 sd-rxRNA sequences.

Table 5 demonstrates inhibition of gene expression with hTGFB1 ori sequences.

Table 6 demonstrates inhibition of gene expression with hTGFB2 ori sequences.

Table 7 demonstrates non-limiting examples of hTGFB2 sd-rxRNA sequences.

Table 8 demonstrates non-limiting examples of hSPP1 sd-rxRNA sequences.

Table 9 demonstrates inhibition of gene expression with hSPP1 ori sequences.

Table 10 demonstrates non-limiting examples of hCTGF sd-rxRNA sequences.

Table 11 demonstrates inhibition of gene expression with hCTGF ori sequences.

Table 12 demonstrates inhibition of gene expression with CTGF ori sequences.

Table 13 demonstrates inhibition of gene expression with SPP1 sd-rxRNA sequences.

Table 14 demonstrates inhibition of gene expression with PTGS2 sd-rxRNA sequences.

Table 15 demonstrates inhibition of gene expression with CTGF sd-rxRNA sequences.

Table 16 demonstrates inhibition of gene expression with TGFB2 sd-rxRNA sequences.

Table 17 demonstrates inhibition of gene expression with TGFB1 sd-rxRNA sequences.

Table 18 demonstrates inhibition of gene expression with SPP1 sd-rxRNA sequences.

Table 19 demonstrates inhibition of gene expression with PTGS2 sd-rxRNA sequences.

Table 20 demonstrates inhibition of gene expression with CTGF sd-rxRNA sequences.

Table 21 demonstrates inhibition of gene expression with TGFB2 sd-rxRNA sequences.

Table 22 demonstrates inhibition of gene expression with TGFB1 sd-rxRNA sequences.

Table 23 provides non-limiting examples of CB1 sequences.

Table 24 provides a summary of CTGF Leads.

Table 25 provides a summary of PTGS2 Leads.

Table 26 provides a summary of TGFβ1 Leads.

Table 27 provides a summary of TGFβ1 Leads.

TABLE 1 Original sd-rxRNA screen Oligo SEQ ID SEQ ID ID# G1- NO Sense-sd-rxRNA GII NO AS-sd-rxRNA-GII 14394 TGFB1 1 GmCmUAAmUGGmUGGAA- 2 5′-P-mU(2′-F-U)(2′-F-C)(2′-F-C)A(2′- chol F-C)(2′-F-C)A(2′-F-U)(2′-F- U)AGmC*A*mC*G*mC*G*G 14395 TGFB1 3 mUGAmUmCGmUGmCGmC 4 5′-P-mGAG(2′-F-C)G(2′-F-C)A(2′-F- mUmC-chol C)GA(2′-F- U)mCA*mU*G*mU*mU*G*G 14396 TGFB1 5 mCAAmUmUmCmCmUGGmC 6 5′-P-mU(2′-F-C)G(2′-F-C)(2′-F- GA-chol C)AGGAA(2′-F- U)mUG*mU*mU*G*mC*mU*G 14397 TGFB1 7 AGmUGGAmUmCmCAmCGA- 8 5′-P-mU(2′-F-C)G(2′-F-U)GGA(2′-F- chol U)(2′-F-C)(2′-F- C)AmCmU*mU*mC*mC*A*G*C 14398 TGFB1 9 mUAmCAGmCAAGGmUmCm 10 5′-P-mGGA(2′-F-C)(2′-F-C)(2′-F- C-chol U)(2′-F-U)G(2′-F-C)(2′-F- U)GmUA*mC*mU*G*mC*G*U 14399 TGFB1 11 AAmCAmUGAmUmCGmUGm 12 5′-P-mG(2′-F-C)A(2′-F-C)GA(2′-F- C-chol U)(2′-F-C)A(2′-F- U)GmUmU*G*G*A*mC*A*G 14400 TGFB1 13 AmCAmUGAmUmCGmUGmC 14 5′-P-mCG(2′-F-C)A(2′-F-C)GA(2′-F- G-chol U)(2′-F-C)A(2′-F- U)GmU*mU*G*G*A*mC*A 14401 TGFB1 15 mCAGmCAAGGmUmCmCmU 16 5′-P-mCAGGA(2′-F-C)(2′-F-C)(2′-F- G-chol U)(2′-F-U)G(2′-F- C)mUG*mU*A*mC*mU*G*C 14402 TGFB1 17 mCmCAAmCAmUGAmUmCG 18 5′-P-mA(2′-F-C)GA(2′-F-U)(2′-F- mU-chol C)A(2′-F-U)G(2′-F-U)(2′-F- U)GG*A*mC*A*G*mC*U 14403 TGFB1 19 AGmCGGAAGmCGmCAmU- 20 5′-P-mA(2′-F-U)G(2′-F-C)G(2′-F- chol C)(2′-F-U)(2′-F-U)(2′-F-C)(2′-F- C)GmCmU*mU*mC*A*mC*mC*A 14404 TGFB1 21 GmCAmUmCGAGGmCmCAm 22 5′-P-mA(2′-F-U)GG(2′-F-C)(2′-F- U-chol C)(2′-F-U)(2′-F-C)GA(2′-F- U)GmC*G*mC*mU*mU*mC*C 14405 TGFB1 23 GAmCmUAmUmCGAmCAmU 24 5′-P-mCA(2′-F-U)G(2′-F-U)(2′-F- G-chol C)GA(2′-F- U)AGmUmC*mU*mU*G*mC*A*G 14406 TGFB1 25 AmCmCmUGmCAAGAmCmU 26 5′-P-mUAG(2′-F-U)(2′-F-C)(2′-F- A-chol U)(2′-F-U)G(2′-F- C)AGGmU*G*G*A*mU*A*G 14407 TGFB1 27 GmCmUmCmCAmCGGAGAA- 28 5′-P-mU(2′-F-U)(2′-F-C)(2′-F-U)(2′-F- chol C)(2′-F-C)G(2′-F- U)GGAGmC*mU*G*A*A*G*C 14408 TGFB2 29 GGmCmUmCmUmCmCmUm 30 5′-P-mU(2′-F- UmCGA-chol C)GAAGGAGAGmCmC*A*mU*mU* mC*G*C 14409 TGFB2 31 GAmCAGGAAmCmCmUGG- 32 5′-P-mC(2′-F-C)AGG(2′-F-U)(2′-F- chol U)(2′-F-C)(2′-F-C)(2′-F- U)GmUmC*mU*mU*mU*A*mU*G 14410 TGFB2 33 mCmCAAGGAGGmUmUmUA- 34 5′-P-mUAAA(2′-F-C)(2′-F-C)(2′-F- chol U)(2′-F-C)(2′-F-C)(2′-F-U)(2′-F- U)GG*mC*G*mU*A*G*U 14411 TGFB2 35 AmUmUmUmCmCAmUmCm 36 5′-P-mUG(2′-F-U)AGA(2′-F- UAmCA-chol U)GGAAAmU*mC*A*mC*mC*mU*C 14412 TGFB2 37 mUmCmCAmUmCmUAmCAA 38 5′-P-mUG(2′-F-U)(2′-F-U)G(2′-F- mCA-chol U)AGA(2′-F- U)GGA*A*A*mU*mC*A*C 14413 TGFB2 39 mUmUmUmCmCAmUmCmU 40 5′-P-mU(2′-F-U)G(2′-F-U)AGA(2′-F- AmCAA-chol U)GGAAA*mU*mC*A*mC*mC*U 14414 TGFB2 41 mCGmCmCAAGGAGGmUmU- 42 5′-P-mAA(2′-F-C)(2′-F-C)(2′-F-U)(2′- chol F-C)(2′-F-C)(2′-F-U)(2′-F- U)GGmCG*mU*A*G*mU*A*C 14415 TGFB2 43 GmUGGmUGAmUmCAGAA- 44 5′-P-mU(2′-F-U)(2′-F-C)(2′-F- chol U)GA(2′-F-U)(2′-F-C)A(2′-F-C)(2′-F- C)AmC*mU*G*G*mU*A*U 14416 TGFB2 45 mCmUmCmCmUGmCmUAA 46 5′-P-mA(2′-F-C)A(2′-F-U)(2′-F- mUGmU-chol U)AG(2′-F- C)AGGAG*A*mU*G*mU*G*G 14417 TGFB2 47 AmCmCmUmCmCAmCAmUA 48 5′-P-mUA(2′-F-U)A(2′-F-U)G(2′-F- mUA-chol U)GGAGGmU*G*mC*mC*A*mU*C 14418 TGFB2 49 AAGmUmCmCAmCmUAGGA- 50 5′-P-mU(2′-F-C)(2′-F-C)(2′-F- chol U)AG(2′-F-U)GGA(2′-F- C)mUmU*mU*A*mU*A*G*U 14419 TGFB2 51 mUGGmUGAmUmCAGAAA- 52 5′-P-mU(2′-F-U)(2′-F-U)(2′-F-C)(2′-F- chol U)GA(2′-F-U)(2′-F-C)A(2′-F- C)mCA*mC*mU*G*G*mU*A 14420 TGFB2 53 AGmUmCmCAmCmUAGGAA- 54 5′-P-mU(2′-F-U)(2′-F-C)(2′-F-C)(2′-F- chol U)AG(2′-F- U)GGAmCmU*mU*mU*A*mU*A*G 14421 TGFB2 55 AmCGmCmCAAGGAGGmU- 56 5′-P-mA(2′-F-C)(2′-F-C)(2′-F-U)(2′-F- chol C)(2′-F-C)(2′-F-U)(2′-F-U)GG(2′-F- C)GmU*A*G*mU*A*mC*U 14422 PTGS2 57 mCAmCAmUmUmUGAmUm 58 5′-P-mU(2′-F-C)AA(2′-F-U)(2′-F- UGA-chol C)AAA(2′-F- U)GmUG*A*mU*mC*mU*G*G 14423 PTGS2 59 mCAmCmUGmCmCmUmCAA 60 5′-P-mAA(2′-F-U)(2′-F-U)GAGG(2′-F- mUmU-chol C)AGmUG*mU*mU*G*A*mU*G 14424 PTGS2 61 AAAmUAmCmCAGmUmCmU 62 5′-P-mAAGA(2′-F-C)(2′-F-U)GG(2′-F- mU-chol U)A(2′-F- U)mUmU*mC*A*mU*mC*mU*G 14425 PTGS2 63 mCAmUmUmUGAmUmUGA 64 5′-P-mUG(2′-F-U)(2′-F-C)AA(2′-F- mCA-chol U)(2′-F- C)AAAmUG*mU*G*A*mU*mC*U

TABLE 2 Inhibition of gene expression with PTGS2 ori sequences Target Gene Gene SEQ ID PTGS2 NM_000963.2 Duplex ID Region Ref Pos No Sense Sequence % Expression (0.1 nM) 15147 CDS 176 65 CCUGGCGCUCAGCCAUACAGCAAAA 59% 15148 CDS 177 66 CUGGCGCUCAGCCAUACAGCAAAUA 72% 15149 CDS 178 67 UGGCGCUCAGCCAUACAGCAAAUCA 77% 15150 CDS 180 68 GCGCUCAGCCAUACAGCAAAUCCUA 70% 15151 CDS 182 69 GCUCAGCCAUACAGCAAAUCCUUGA 76% 15152 CDS 183 70 CUCAGCCAUACAGCAAAUCCUUGCA 74% 15153 CDS 184 71 UCAGCCAUACAGCAAAUCCUUGCUA 47% 15154 CDS 186 72 AGCCAUACAGCAAAUCCUUGCUGUA 55% 15155 CDS 187 73 GCCAUACAGCAAAUCCUUGCUGUUA 41% 15156 CDS 188 74 CCAUACAGCAAAUCCUUGCUGUUCA 46% 15157 CDS 212 75 CCACCCAUGUCAAAACCGAGGUGUA 31% 15158 CDS 243 76 AGUGUGGGAUUUGACCAGUAUAA 23% GA 15159 CDS 244 77 GUGUGGGAUUUGACCAGUAUAAG 24% UA 15160 CDS 245 78 UGUGGGAUUUGACCAGUAUAAGU 38% GA 15161 CDS 252 79 UUUGACCAGUAUAAGUGCGAUUG 29% UA 15162 CDS 285 80 GGAUUCUAUGGAGAAAACUGCUCAA 16% 15163 CDS 337 81 UAUUUCUGAAACCCACUCCAAACAA 32% 15164 CDS 338 82 AUUUCUGAAACCCACUCCAAACACA 21% 15165 CDS 339 83 UUUCUGAAACCCACUCCAAACACAA 21% 15166 CDS 340 84 UUCUGAAACCCACUCCAAACACAGA 45% 15167 CDS 345 85 AAACCCACUCCAAACACAGUGCACA 87% 15168 CDS 347 86 ACCCACUCCAAACACAGUGCACUAA 83% 15169 CDS 349 87 CCACUCCAAACACAGUGCACUACAA 51% 15170 CDS 350 88 CACUCCAAACACAGUGCACUACAUA 31% 15171 CDS 394 89 UUUGGAACGUUGUGAAUAACAUU 43% CA 15172 CDS 406 90 UGAAUAACAUUCCCUUCCUUCGAAA 21% 15173 CDS 407 91 GAAUAACAUUCCCUUCCUUCGAAAA 32% 15174 CDS 408 92 AAUAACAUUCCCUUCCUUCGAAAUA 27% 15175 CDS 435 93 AUUAUGAGUUAUGUGUUGACAUC 27% CA 15176 CDS 437 94 UAUGAGUUAUGUGUUGACAUCCA 21% GA 15177 CDS 439 95 UGAGUUAUGUGUUGACAUCCAGA 30% UA 15178 CDS 440 96 GAGUUAUGUGUUGACAUCCAGAU 68% CA 15179 CDS 441 97 AGUUAUGUGUUGACAUCCAGAUCAA 35% 15180 CDS 442 98 GUUAUGUGUUGACAUCCAGAUCACA 36% 15181 CDS 443 99 UUAUGUGUUGACAUCCAGAUCACAA 51% 15182 CDS 444 100 UAUGUGUUGACAUCCAGAUCACAUA 24% 15183 CDS 445 101 AUGUGUUGACAUCCAGAUCACAUUA 37% 15184 CDS 446 102 UGUGUUGACAUCCAGAUCACAUUUA 42% 15185 CDS 448 103 UGUUGACAUCCAGAUCACAUUUGAA 18% 15186 CDS 449 104 GUUGACAUCCAGAUCACAUUUGAUA 24% 15187 CDS 450 105 UUGACAUCCAGAUCACAUUUGAUUA 25% 15188 CDS 452 106 GACAUCCAGAUCACAUUUGAUUGAA 27% 15189 CDS 454 107 CAUCCAGAUCACAUUUGAUUGACAA 27% 15190 CDS 455 108 AUCCAGAUCACAUUUGAUUGACAGA 32% 15191 CDS 456 109 UCCAGAUCACAUUUGAUUGACAGUA 40% 15192 CDS 457 110 CCAGAUCACAUUUGAUUGACAGUCA 52% 15193 CDS 460 111 GAUCACAUUUGAUUGACAGUCCACA 40% 15194 CDS 461 112 AUCACAUUUGAUUGACAGUCCACCA 46% 15195 CDS 462 113 UCACAUUUGAUUGACAGUCCACCAA 34% 15196 CDS 463 114 CACAUUUGAUUGACAGUCCACCAAA 30% 15197 CDS 464 115 ACAUUUGAUUGACAGUCCACCAACA 32% 15198 CDS 465 116 CAUUUGAUUGACAGUCCACCAACUA 44% 15199 CDS 467 117 UUUGAUUGACAGUCCACCAACUUAA 17% 15200 CDS 468 118 UUGAUUGACAGUCCACCAACUUACA 22% 15201 CDS 469 119 UGAUUGACAGUCCACCAACUUACAA 27% 15202 CDS 470 120 GAUUGACAGUCCACCAACUUACAAA 41% 15203 CDS 471 121 AUUGACAGUCCACCAACUUACAAUA 39% 15204 CDS 472 122 UUGACAGUCCACCAACUUACAAUGA 61% 15205 CDS 473 123 UGACAGUCCACCAACUUACAAUGCA 48% 15206 CDS 479 124 UCCACCAACUUACAAUGCUGACUAA 29% 15207 CDS 486 125 ACUUACAAUGCUGACUAUGGCUACA 35% 15208 CDS 488 126 UUACAAUGCUGACUAUGGCUACAAA 32% 15209 CDS 517 127 GGGAAGCCUUCUCUAACCUCUCCUA 39% 15210 CDS 518 128 GGAAGCCUUCUCUAACCUCUCCUAA 48% 15211 CDS 519 129 GAAGCCUUCUCUAACCUCUCCUAUA 19% 15212 CDS 520 130 AAGCCUUCUCUAACCUCUCCUAUUA 17% 15213 CDS 524 131 CUUCUCUAACCUCUCCUAUUAUACA 17% 15214 CDS 525 132 UUCUCUAACCUCUCCUAUUAUACUA 34% 15215 CDS 526 133 UCUCUAACCUCUCCUAUUAUACUAA 49% 15216 CDS 601 134 GUAAAAAGCAGCUUCCUGAUUCAAA 35% 15217 CDS 602 135 UAAAAAGCAGCUUCCUGAUUCAAAA 25% 15218 CDS 606 136 AAGCAGCUUCCUGAUUCAAAUGAGA 27% 15219 CDS 615 137 CCUGAUUCAAAUGAGAUUGUGGAAA 37% 15220 CDS 616 138 CUGAUUCAAAUGAGAUUGUGGAA 27% AA 15221 CDS 636 139 GAAAAAUUGCUUCUAAGAAGAAAGA 37% 15222 CDS 637 140 AAAAAUUGCUUCUAAGAAGAAAGUA 56% 15223 CDS 638 141 AAAAUUGCUUCUAAGAAGAAAGUUA 25% 15224 CDS 639 142 AAAUUGCUUCUAAGAAGAAAGUUCA 34% 15225 CDS 678 143 GGCUCAAACAUGAUGUUUGCAUUCA 68% 15226 CDS 679 144 GCUCAAACAUGAUGUUUGCAUUCUA 51% 15227 CDS 680 145 CUCAAACAUGAUGUUUGCAUUCUUA 50% 15228 CDS 682 146 CAAACAUGAUGUUUGCAUUCUUU 51% GA 15229 CDS 683 147 AAACAUGAUGUUUGCAUUCUUUG 63% CA 15230 CDS 722 148 UCAGUUUUUCAAGACAGAUCAUAAA 45% 15231 CDS 723 149 CAGUUUUUCAAGACAGAUCAUAAGA 59% 15232 CDS 724 150 AGUUUUUCAAGACAGAUCAUAAGCA 80% 15233 CDS 725 151 GUUUUUCAAGACAGAUCAUAAGCGA 55% 15234 CDS 726 152 UUUUUCAAGACAGAUCAUAAGCGAA 53% 15235 CDS 776 153 CCAUGGGGUGGACUUAAAUCAUAUA 56% 15236 CDS 787 154 ACUUAAAUCAUAUUUACGGUGAAAA 63% 15237 CDS 788 155 CUUAAAUCAUAUUUACGGUGAAACA 43% 15238 CDS 789 156 UUAAAUCAUAUUUACGGUGAAACUA 48% 15239 CDS 790 157 UAAAUCAUAUUUACGGUGAAACUCA 56% 15240 CDS 792 158 AAUCAUAUUUACGGUGAAACUCUGA 46% 15241 CDS 793 159 AUCAUAUUUACGGUGAAACUCUGGA 64% 15242 CDS 799 160 UUUACGGUGAAACUCUGGCUAGACA 35% 15243 CDS 819 161 AGACAGCGUAAACUGCGCCUUUUCA 65% 15244 CDS 820 162 GACAGCGUAAACUGCGCCUUUUCAA 51% 15245 CDS 821 163 ACAGCGUAAACUGCGCCUUUUCAAA 48% 15246 CDS 822 164 CAGCGUAAACUGCGCCUUUUCAAGA 61% 15247 CDS 823 165 AGCGUAAACUGCGCCUUUUCAAGGA 48% 15248 CDS 828 166 AAACUGCGCCUUUUCAAGGAUGGAA 42% 15249 CDS 830 167 ACUGCGCCUUUUCAAGGAUGGAAAA 29% 15250 CDS 861 168 UAUCAGAUAAUUGAUGGAGAGAU 32% GA 15251 CDS 862 169 AUCAGAUAAUUGAUGGAGAGAUG 55% UA 15252 CDS 863 170 UCAGAUAAUUGAUGGAGAGAUGU 50% AA 15253 CDS 864 171 CAGAUAAUUGAUGGAGAGAUGUA 50% UA 15254 CDS 865 172 AGAUAAUUGAUGGAGAGAUGUAU 55% CA 15255 CDS 866 173 GAUAAUUGAUGGAGAGAUGUAUC 65% CA 15256 CDS 867 174 AUAAUUGAUGGAGAGAUGUAUCC 54% UA 15257 CDS 880 175 AGAUGUAUCCUCCCACAGUCAAAGA 78% 15258 CDS 881 176 GAUGUAUCCUCCCACAGUCAAAGAA 79% 15259 CDS 882 177 AUGUAUCCUCCCACAGUCAAAGAUA 49% 15260 CDS 883 178 UGUAUCCUCCCACAGUCAAAGAUAA 28% 15261 CDS 884 179 GUAUCCUCCCACAGUCAAAGAUACA 56% 15262 CDS 885 180 UAUCCUCCCACAGUCAAAGAUACUA 42% 15263 CDS 887 181 UCCUCCCACAGUCAAAGAUACUCAA 45% 15264 CDS 888 182 CCUCCCACAGUCAAAGAUACUCAGA 73% 15265 CDS 889 183 CUCCCACAGUCAAAGAUACUCAGGA 56% 15266 CDS 891 184 CCCACAGUCAAAGAUACUCAGGCAA 80% 15267 CDS 901 185 AAGAUACUCAGGCAGAGAUGAUCUA 59% 15268 CDS 935 186 AGUCCCUGAGCAUCUACGGUUUGCA 83% 15269 CDS 980 187 UCUGGUGCCUGGUCUGAUGAUGU 55% AA 15270 CDS 981 188 CUGGUGCCUGGUCUGAUGAUGUA 56% UA 15271 CDS 982 189 UGGUGCCUGGUCUGAUGAUGUAU 43% GA 15272 CDS 983 190 GGUGCCUGGUCUGAUGAUGUAUG 41% CA 15273 CDS 984 191 GUGCCUGGUCUGAUGAUGUAUGC 42% CA 15274 CDS 985 192 UGCCUGGUCUGAUGAUGUAUGCC 37% AA 15275 CDS 986 193 GCCUGGUCUGAUGAUGUAUGCCACA 61% 15276 CDS 1016 194 GCUGCGGGAACACAACAGAGUAUGA 44% 15277 CDS 1019 195 GCGGGAACACAACAGAGUAUGCGAA 33% 15278 CDS 1038 196 UGCGAUGUGCUUAAACAGGAGCAUA 53% 15279 CDS 1039 197 GCGAUGUGCUUAAACAGGAGCAUCA 109% 15280 CDS 1040 198 CGAUGUGCUUAAACAGGAGCAUCCA 77% 15281 CDS 1042 199 AUGUGCUUAAACAGGAGCAUCCUGA 69% 15282 CDS 1043 200 UGUGCUUAAACAGGAGCAUCCUGAA 76% 15283 CDS 1044 201 GUGCUUAAACAGGAGCAUCCUGAAA 65% 15284 CDS 1045 202 UGCUUAAACAGGAGCAUCCUGAAUA 64% 15285 CDS 1084 203 UGUUCCAGACAAGCAGGCUAAUACA 41% 15286 CDS 1093 204 CAAGCAGGCUAAUACUGAUAGGAGA 24% 15287 CDS 1095 205 AGCAGGCUAAUACUGAUAGGAGAGA 50% 15288 CDS 1096 206 GCAGGCUAAUACUGAUAGGAGAGAA 51% 15289 CDS 1124 207 UAAGAUUGUGAUUGAAGAUUAUG 35% UA 15290 CDS 1125 208 AAGAUUGUGAUUGAAGAUUAUGU 34% GA 15291 CDS 1126 209 AGAUUGUGAUUGAAGAUUAUGUG 59% CA 15292 CDS 1127 210 GAUUGUGAUUGAAGAUUAUGUGC 41% AA 15293 CDS 1128 211 AUUGUGAUUGAAGAUUAUGUGCA 51% AA 15294 CDS 1129 212 UUGUGAUUGAAGAUUAUGUGCAA 45% CA 15295 CDS 1131 213 GUGAUUGAAGAUUAUGUGCAACACA 37% 15296 CDS 1132 214 UGAUUGAAGAUUAUGUGCAACACUA 34% 15297 CDS 1134 215 AUUGAAGAUUAUGUGCAACACUUGA 24% 15298 CDS 1136 216 UGAAGAUUAUGUGCAACACUUGAGA 37% 15299 CDS 1138 217 AAGAUUAUGUGCAACACUUGAGUGA 44% 15300 CDS 1145 218 UGUGCAACACUUGAGUGGCUAUCAA 29% 15301 CDS 1149 219 CAACACUUGAGUGGCUAUCACUUCA 33% 15302 CDS 1179 220 AAAUUUGACCCAGAACUACUUUUCA 35% 15303 CDS 1180 221 AAUUUGACCCAGAACUACUUUUCAA 41% 15304 CDS 1181 222 AUUUGACCCAGAACUACUUUUCAAA 40% 15305 CDS 1200 223 UUCAACAAACAAUUCCAGUACCAAA 49% 15306 CDS 1211 224 AUUCCAGUACCAAAAUCGUAUUGCA 27% 15307 CDS 1217 225 GUACCAAAAUCGUAUUGCUGCUGAA 31% 15308 CDS 1270 226 UUCUGCCUGACACCUUUCAAAUUCA 35% 15309 CDS 1280 227 CACCUUUCAAAUUCAUGACCAGAAA 57% 15310 CDS 1284 228 UUUCAAAUUCAUGACCAGAAAUACA 42% 15311 CDS 1289 229 AAUUCAUGACCAGAAAUACAACUAA 52% 15312 CDS 1327 230 ACAACAACUCUAUAUUGCUGGAACA 58% 15313 CDS 1352 231 UGGAAUUACCCAGUUUGUUGAAU 35% CA 15314 CDS 1356 232 AUUACCCAGUUUGUUGAAUCAUUCA 41% 15315 CDS 1357 233 UUACCCAGUUUGUUGAAUCAUUCAA 58% 15316 CDS 1359 234 ACCCAGUUUGUUGAAUCAUUCACCA 52% 15317 CDS 1360 235 CCCAGUUUGUUGAAUCAUUCACCAA 66% 15318 CDS 1361 236 CCAGUUUGUUGAAUCAUUCACCAGA 54% 15319 CDS 1365 237 UUUGUUGAAUCAUUCACCAGGCAAA 47% 15320 CDS 1462 238 AGAGCAGGCAGAUGAAAUACCAGUA 65% 15321 CDS 1463 239 GAGCAGGCAGAUGAAAUACCAGUCA 66% 15322 CDS 1465 240 GCAGGCAGAUGAAAUACCAGUCUUA 22% 15323 CDS 1466 241 CAGGCAGAUGAAAUACCAGUCUUUA 43% 15324 CDS 1472 242 GAUGAAAUACCAGUCUUUUAAUGAA 23% 15325 CDS 1473 243 AUGAAAUACCAGUCUUUUAAUGAGA 61% 15326 CDS 1474 244 UGAAAUACCAGUCUUUUAAUGAG 49% UA 15327 CDS 1475 245 GAAAUACCAGUCUUUUAAUGAGUAA 76% 15328 CDS 1476 246 AAAUACCAGUCUUUUAAUGAGUACA 51% 15329 CDS 1477 247 AAUACCAGUCUUUUAAUGAGUACCA 72% 15330 CDS 1478 248 AUACCAGUCUUUUAAUGAGUACCGA 40% 15331 CDS 1479 249 UACCAGUCUUUUAAUGAGUACCGCA 53% 15332 CDS 1480 250 ACCAGUCUUUUAAUGAGUACCGCAA 39% 15333 CDS 1481 251 CCAGUCUUUUAAUGAGUACCGCAAA 41% 15334 CDS 1483 252 AGUCUUUUAAUGAGUACCGCAAACA 38% 15335 CDS 1485 253 UCUUUUAAUGAGUACCGCAAACGCA 55% 15336 CDS 1486 254 CUUUUAAUGAGUACCGCAAACGCUA 63% 15337 CDS 1487 255 UUUUAAUGAGUACCGCAAACGCUUA 52% 15338 CDS 1495 256 AGUACCGCAAACGCUUUAUGCUGAA 49% 15339 CDS 1524 257 UAUGAAUCAUUUGAAGAACUUACAA 65% 15340 CDS 1525 258 AUGAAUCAUUUGAAGAACUUACAGA 63% 15341 CDS 1527 259 GAAUCAUUUGAAGAACUUACAGGAA 65% 15342 CDS 1529 260 AUCAUUUGAAGAACUUACAGGAGAA 43% 15343 CDS 1531 261 CAUUUGAAGAACUUACAGGAGAAAA 63% 15344 CDS 1532 262 AUUUGAAGAACUUACAGGAGAAAAA 33% 15345 CDS 1574 263 GGAAGCACUCUAUGGUGACAUCGAA 62% 15346 CDS 1609 264 UGUAUCCUGCCCUUCUGGUAGAAAA 36% 15347 CDS 1614 265 CCUGCCCUUCUGGUAGAAAAGCCUA 58% 15348 CDS 1650 266 AUCUUUGGUGAAACCAUGGUAGAAA 60% 15349 CDS 1666 267 UGGUAGAAGUUGGAGCACCAUUCUA 88% 15350 CDS 1669 268 UAGAAGUUGGAGCACCAUUCUCCUA 85% 15351 CDS 1672 269 AAGUUGGAGCACCAUUCUCCUUGAA 83% 15352 CDS 1675 270 UUGGAGCACCAUUCUCCUUGAAAGA 85% 15353 CDS 1676 271 UGGAGCACCAUUCUCCUUGAAAGGA 83% 15354 CDS 1677 272 GGAGCACCAUUCUCCUUGAAAGGAA 74% 15355 CDS 1678 273 GAGCACCAUUCUCCUUGAAAGGACA 81% 15356 CDS 1679 274 AGCACCAUUCUCCUUGAAAGGACUA 86% 15357 CDS 1680 275 GCACCAUUCUCCUUGAAAGGACUUA 98% 15358 CDS 1681 276 CACCAUUCUCCUUGAAAGGACUUAA 78% 15359 CDS 1682 277 ACCAUUCUCCUUGAAAGGACUUAUA 88% 15360 CDS 1683 278 CCAUUCUCCUUGAAAGGACUUAUGA 88% 15361 CDS 1762 279 UGGGUUUUCAAAUCAUCAACACUGA 78% 15362 CDS 1763 280 GGGUUUUCAAAUCAUCAACACUGCA 92% 15363 CDS 1767 281 UUUCAAAUCAUCAACACUGCCUCAA 85% 15364 CDS 1770 282 CAAAUCAUCAACACUGCCUCAAUUA 84% 15365 CDS 1773 283 AUCAUCAACACUGCCUCAAUUCAGA 86% 15366 CDS 1774 284 UCAUCAACACUGCCUCAAUUCAGUA 94% 15367 CDS 1775 285 CAUCAACACUGCCUCAAUUCAGUCA 84% 15368 CDS 1776 286 AUCAACACUGCCUCAAUUCAGUCUA 84% 15369 CDS 1777 287 UCAACACUGCCUCAAUUCAGUCUCA 68% 15370 CDS 1778 288 CAACACUGCCUCAAUUCAGUCUCUA 73% 15371 CDS 1779 289 AACACUGCCUCAAUUCAGUCUCUCA 79% 15372 CDS 1780 290 ACACUGCCUCAAUUCAGUCUCUCAA 78% 15373 CDS 1781 291 CACUGCCUCAAUUCAGUCUCUCAUA 92% 15374 CDS 1782 292 ACUGCCUCAAUUCAGUCUCUCAUCA 89% 15375 CDS 1783 293 CUGCCUCAAUUCAGUCUCUCAUCUA 95% 15376 CDS 1784 294 UGCCUCAAUUCAGUCUCUCAUCUGA 83% 15377 CDS 1785 295 GCCUCAAUUCAGUCUCUCAUCUGCA 46% 15378 CDS 1786 296 CCUCAAUUCAGUCUCUCAUCUGCAA 51% 15379 CDS 1787 297 CUCAAUUCAGUCUCUCAUCUGCAAA 61% 15380 CDS 1790 298 AAUUCAGUCUCUCAUCUGCAAUAAA 30% 15381 CDS 1791 299 AUUCAGUCUCUCAUCUGCAAUAACA 32% 15382 CDS 1792 300 UUCAGUCUCUCAUCUGCAAUAACGA 30% 15383 CDS 1793 301 UCAGUCUCUCAUCUGCAAUAACGUA 38% 15384 CDS 1794 302 CAGUCUCUCAUCUGCAAUAACGUGA 67% 15385 CDS 1795 303 AGUCUCUCAUCUGCAAUAACGUGAA 71% 15386 CDS 1796 304 GUCUCUCAUCUGCAAUAACGUGAAA 81% 15387 CDS 1856 305 AGAGCUCAUUAAAACAGUCACCAUA 33% 15388 CDS 1857 306 GAGCUCAUUAAAACAGUCACCAUCA 55% 15389 CDS 1858 307 AGCUCAUUAAAACAGUCACCAUCAA 31% 15390 CDS 1859 308 GCUCAUUAAAACAGUCACCAUCAAA 46% 15391 CDS 1860 309 CUCAUUAAAACAGUCACCAUCAAUA 43% 15392 CDS 1861 310 UCAUUAAAACAGUCACCAUCAAUGA 58% 15393 CDS 1862 311 CAUUAAAACAGUCACCAUCAAUGCA 78% 15394 CDS 1864 312 UUAAAACAGUCACCAUCAAUGCAAA 41% 15395 CDS 1865 313 UAAAACAGUCACCAUCAAUGCAAGA 80% 15396 CDS 1866 314 AAAACAGUCACCAUCAAUGCAAGUA 79% 15397 CDS 1868 315 AACAGUCACCAUCAAUGCAAGUUCA 34% 15398 CDS 1912 316 AUAUCAAUCCCACAGUACUACUAAA 39% 15399 CDS 1928 317 ACUACUAAAAGAACGUUCGACUGAA 39% 15400 CDS/3UTR 1941 318 CGUUCGACUGAACUGUAGAAGUCUA 30% 15401 CDS/3UTR 1946 319 GACUGAACUGUAGAAGUCUAAUGAA 25% 15402 CDS/3UTR 1949 320 UGAACUGUAGAAGUCUAAUGAUCAA 29% 15403 3UTR 2077 321 UCCUGUUGCGGAGAAAGGAGUCAUA 45% 15404 3UTR 2082 322 UUGCGGAGAAAGGAGUCAUACUU 43% GA 15405 3UTR 2098 323 CAUACUUGUGAAGACUUUUAUGU 30% CA 15406 3UTR 2128 324 CUAAAGAUUUUGCUGUUGCUGUU 41% AA 15407 3UTR 2141 325 UGUUGCUGUUAAGUUUGGAAAAC 29% AA 15408 3UTR 2188 326 AGAGAGAAAUGAGUUUUGACGUC 26% UA 15409 3UTR 2235 327 UUAUAAGAACGAAAGUAAAGAUGUA 33% 15410 3UTR 2281 328 AAGAUGGCAAAAUGCUGAAAGUUUA 28% 15411 3UTR 2305 329 UUACACUGUCGAUGUUUCCAAUGCA 46% 15412 3UTR 2446 330 GACAUUACCAGUAAUUUCAUGUCUA 24% 15413 3UTR 2581 331 CAAAAAGAAGCUGUCUUGGAUUUAA 36% 15414 3UTR 2669 332 CUUUUUCACCAAGAGUAUAAACCUA 41% 15415 3UTR 2730 333 AUGCCAAAUUUAUUAAGGUGGUG 61% GA 15416 3UTR 2750 334 GUGGAGCCACUGCAGUGUUAUCU 39% UA 15417 3UTR 2752 335 GGAGCCACUGCAGUGUUAUCUUAAA 45% 15418 3UTR 2802 336 CAGAAUUUGUUUAUAUGGCUGGU 49% AA 15419 3UTR 2810 337 GUUUAUAUGGCUGGUAACAUGUA 34% AA 15420 3UTR 2963 338 UACUCAGAUUUUGCUAUGAGGUU 42% AA 15421 3UTR 2967 339 CAGAUUUUGCUAUGAGGUUAAUG 39% AA 15422 3UTR 2970 340 AUUUUGCUAUGAGGUUAAUGAAG 43% UA 15423 3UTR 2986 341 AAUGAAGUACCAAGCUGUGCUUGAA 40% 15424 3UTR 3064 342 AUCACAUUGCAAAAGUAGCAAUGAA 59% 15425 3UTR 3072 343 GCAAAAGUAGCAAUGACCUCAUAAA 35% 15426 3UTR 3083 344 AAUGACCUCAUAAAAUACCUCUUCA 40% 15427 3UTR 3134 345 AAUUUUAUCUCAGUCUUGAAGCCAA 55% 15428 3UTR 3147 346 UCUUGAAGCCAAUUCAGUAGGUGCA 52% 15429 3UTR 3157 347 AAUUCAGUAGGUGCAUUGGAAUCAA 71% 15430 3UTR 3212 348 UUUCUUCUUUUAGCCAUUUUGCU 38% AA 15431 3UTR 3216 349 UUCUUUUAGCCAUUUUGCUAAGA 40% GA 15432 3UTR 3225 350 CCAUUUUGCUAAGAGACACAGUCUA 36% 15433 3UTR 3278 351 UUACUAGUUUUAAGAUCAGAGUU 70% CA 15434 3UTR 3313 352 ACUCUGCCUAUAUUUUCUUACCUGA 56% 15435 3UTR 3335 353 UGAACUUUUGCAAGUUUUCAGGU 64% AA 15436 3UTR 3336 354 GAACUUUUGCAAGUUUUCAGGUA 62% AA 15437 3UTR 3351 355 UUCAGGUAAACCUCAGCUCAGGACA 62% 15438 3UTR 3360 356 ACCUCAGCUCAGGACUGCUAUUUAA 53% 15439 3UTR 3441 357 CUUAUUUUAAGUGAAAAGCAGAGAA 83% 15440 3UTR 3489 358 UAUCUGUAACCAAGAUGGAUGCAAA 93% 15441 3UTR 3662 359 UUUUCCACAUCUCAUUGUCACUGAA 36% 15442 3UTR 3668 360 ACAUCUCAUUGUCACUGACAUUUAA 40% 15443 3UTR 3735 361 GUCUUAUUAGGACACUAUGGUUA 40% UA 15444 3UTR 3737 362 CUUAUUAGGACACUAUGGUUAUA 37% AA 15445 3UTR 3738 363 UUAUUAGGACACUAUGGUUAUAA 38% AA 15446 3UTR 3752 364 UGGUUAUAAACUGUGUUUAAGCC 28% UA 15447 3UTR 3919 365 AUAUUUAAGGUUGAAUGUUUGUC 40% CA 15448 3UTR 3961 366 CUAGCCCACAAAGAAUAUUGUCUCA 47% 15449 3UTR 3981 367 UCUCAUUAGCCUGAAUGUGCCAUAA 56% 15450 3UTR 3994 368 AAUGUGCCAUAAGACUGACCUUUUA 52%

TABLE 3 PTGS2 sd-rxRNA Duplex ID ID Sequence SEQ ID NO 17388 17062 G.A.A.A.A.mC.mU.G.mC.mU.mC.A.A.Chl 369 17063 P.mU.fU.G.A.G.fC.A.G.fU.fU.fU.fU.fC*fU*fC*fC*A*fU*A 370 17389 17064 A.mC.mC.mU.mC.mU.mC.mC.mU.A.mU.mU.A.Chl 371 17065 P.mU.A.A.fU.A.G.G.A.G.A.G.G.fU*fU*A*G*A*G*A 372 17390 17066 mU.mC.mC.A.mC.mC.A.A.mC.mU.mU.A.A.Chl 373 17068 P.mU.fU.A.A.G.fU.fU.G.G.fU.G.G.A*fC*fU*G*fU*fC*A 374 17391 17067 G.mU.mC.mC.A.mC.mC.A.A.mC.mU.mU.A.A.Chl 375 17068 P.mU.fU.A.A.G.fU.fU.G.G.fU.G.G.A*fC*fU*G*fU*fC*A 376 17392 17069 mC.mU.mC.mC.mU.A.mU.mU.A.mU.A.mC.A.Chl 377 17070 P.mU.G.fU.A.fU.A.A.fU.A.G.G.A.G*A*G*G*fU*fU*A 378 17393 17071 G.A.mU.mC.A.mC.A.mU.mU.mU.G.A.A.Chl 379 17073 P.mU.fU.fC.A.A.A.fU.G.fU.G.A.fU.fC*fU*G*G*A*fU*G 380 17394 17072 A.G.A.mU.mC.A.mC.A.mU.mU.mU.G.A.A.Chl 381 17073 P.mU.fU.fC.A.A.A.fU.G.fU.G.A.fU.fC*fU*G*G*A*fU*G 382 17395 17074 A.A.mC.mC.mU.mC.mU.mC.mC.mU.A.mU.A.Chl 383 17075 P.mU.A.fU.A.G.G.A.G.A.G.G.fU.fU*A*G*A*G*A*A 384 17396 17076 G.mU.mU.G.A.mC.A.mU.mC.mC.A.G.A.Chl 385 17077 P.mU.fC.fU.G.G.A.fU.G.fU.fC.A.A.fC*A*fC*A*fU*A*A 386 17397 17078 mC.mC.mU.mU.mC.mC.mU.mU.mC.G.A.A.A.Chl 387 17079 P.mU.fU.fU.fC.G.A.A.G.G.A.A.G.G*G*A*A*fU*G*U 388 17398 17080 A.mC.mU.mC.mC.A.A.A.mC.A.mC.A.A.Chl 389 17082 P.mU.fU.G.fU.G.fU.fU.fU.G.G.A.G.fU*G*G*G*fU*fU*U 390 17399 17081 mC.A.mC.mU.mC.mC.A.A.A.mC.A.mC.A.A.Chl 391 17082 P.mU.fU.G.fU.G.fU.fU.fU.G.G.A.G.fU*G*G*G*fU*fU*U 392 17400 17083 mC.A.mC.mU.mC.mC.A.A.A.mC.A.mC.A.Chl 393 17084 P.mUGfUGfUfUfUGGAGfUG*G*G*fU*fU*fU*C 394 17401 17085 mC.mC.A.mC.mC.A.A.mC.mU.mU.A.mCA.Chl 395 17087 P.mUGfUAAGfUfUGGfUGG*A*fC*fU*G*fU*C 396 17402 17086 mU.mC.mC.A.mC.mC.A.A.mC.mU.mU.A.mCA.Chl 397 17087 P.mUGfUAAGfUfUGGfUGG*A*fC*fU*G*fU*C 398 17403 17088 A.A.mU.A.mC.mC.A.G.mU.mC.mU.mU.A.Chl 399 17089 P.mU.A.A.G.A.fC.fU.G.G.fU.A.fU.fU*fU*fC*A*fU*fC*U 400 17404 17090 G.A.mC.mC.A.G.mU.A.mU.A.A.G.A.Chl 401 17091 P.mU.fC.fU.fU.A.fU.A.fC.fU.G.G.fU.fC*A*A*A*fU*fC*C 402 17405 17092 G.mU.mC.mU.mU.mU.mU.A.A.mU.G.A.A.Chl 403 17093 P.mU.fU.fC.A.fU.fU.A.A.A.A.G.A.fC*fU*G*G*fU*A*U 404 17406 17094 A.A.mU.mU.mU.mC.A.mU.G.mU.mC.mU.A.Chl 405 17095 P.mU.A.G.A.fC.A.fU.G.A.A.A.fU.fU*A*fC*fU*G*G*U 406 17407 17096 A.mU.mC.A.mC.A.mU.mU.mU.G.A.mU.A.Chl 407 17098 P.mU.A.fU.fC.A.A.A.fU.G.fU.G.A.fU*fC*fU*G*G*A*U 408 17408 17097 G.A.mU.mC.A.mC.A.mU.mU.mU.G.A.mU.A.Chl 409 17098 P.mU.A.fU.fC.A.A.A.fU.G.fU.G.A.fU*fC*fU*G*G*A*U 410 17409 17099 mU.mC.mC.A.G.A.mU.mC.A.mC.A.mU.A.Chl 411 17100 P.mU.A.fU.G.fU.G.A.fU.fC.fU.G.G.A*fU*G*fU*fC*A*A 412 17410 17101 mU.A.mC.mU.G.A.mU.A.G.G.A.G.A.Chl 413 17102 P.mU.fC.fU.fC.fC.fU.A.fU.fC.A.G.fU.A*fU*fU*A*G*fC*C 414 17411 17103 G.mU.G.mC.A.A.mC.A.mC.mU.fU.G.A.Chl 415 17104 P.mU.fC.A.A.G.fU.G.fU.fU.G.mC.A.fC*A*fU*A*A*fU*C 416 17412 17105 A.mC.mC.A.G.mU.A.mU.A.A.G.mU.A.Chl 417 17106 P.mU.A.fC.fU.fU.A.fU.A.fC.fU.G.G.fU*fC*A*A*A*fU*C 418 17413 17107 G.A.A.G.mU.mC.mU.A.A.mU.G.A.A.Chl 419 17108 P.mU.fU.fC.A.fU.fU.A.G.A.fC.mU.fU.fC*fU*A*fC*A*G*U 420 17414 17109 A.A.G.A.A.G.A.A.A.G.mU.mU.A.Chl 421 17110 P.mU.A.A.fC.fU.fU.fU.fC.fU.fU.fC.fU.fU*A*G*A*A*G*C 422 17415 17111 mU.mC.A.mC.A.mU.mU.mU.G.AmU.mU.A.Chl 423 17113 P.mU.A.A.fU.fC.A.A.A.fU.G.fUG.A*fU*fC*fU*G*G*A 424 17416 17112 A.mU.mC.A.mC.A.mU.mU.mU.G.AmU.mU.A.Chl 425 17113 P.mU.A.A.fU.fC.A.A.A.fU.G.fUG.A*fU*fC*fU*G*G*A 426 17417 17114 A.mC.A.mU.mU.mU.G.A.mU.mUG.A.A.Chl 427 17116 P.mU.fU.fC.A.A.fU.fC.A.A.A.fUG.fU*G*A*fU*fC*fU*G 428 17418 17115 mC.A.mC.A.mU.mU.mU.G.A.mU.mUG.A.A.Chl 429 17116 P.mU.fU.fC.A.A.fU.fC.A.A.A.fUG.fU*G*A*fU*fC*fU*G 430 17419 17117 A.mU.mU.mU.G.A.mU.mU.G.AmC.A.A.Chl 431 17119 P.mU.fU.G.fU.fC.A.A.fU.fC.A.AA.fU*G*fU*G*A*fU*C 432 17420 17118 mC.A.mU.mU.mU.G.A.mU.mU.G.AmC.A.A.Chl 433 17119 P.mU.fU.G.fU.fC.A.A.fU.fC.A.AA.fU*G*fU*G*A*fU*C 434 17421 17120 mC.A.mU.mC.mU.G.mC.A.A.mU.A.A.A.Chl 435 17122 P.mU.fU.fU.A.fU.fU.G.fC.A.G.A.fU.G*A*G*A*G*A*C 436 17422 17121 mU.mC.A.mU.mC.mU.G.mC.A.A.mU.A.A.A.Chl 437 17122 P.mU.fU.fU.A.fU.fU.G.fC.A.G.A.fU.G*A*G*A*G*A*C 438

TABLE 4 TGFB1 sd-rxRNA Target Gene hTGFB1 Duplex ID Single Strand ID sd-rxRNA sequence SEQ ID NO 18454 17491 mC.A.mC.A.G.mC.A.mU.A.mU.A.mU.A.Chl 439 17492 P.mU.A.fU.A.fU.A.fU.G.fC.fU.G.fU.G*fU*G*fU*A*fC*U 440 18455 17493 mC.A.G.mC.A.mU.A.mU.A.mU.A.mU.A.Chl 441 17494 P.mU.A.fU.A.fU.A.fU.A.fU.G.fC.fU.G*fU*G*fU*G*fU*A 442 18456 17495 G.mU.A.mC.A.mU.mU.G.A.mC.mU.mU.A.Chl 443 17497 P.mU.A.A.G.fU.fC.A.A.fU.G.fU.A.fC*A*G*fC*fU*G*C 444 18457 17496 mU.G.mU.A.mC.A.mU.mU.G.A.mC.mU.mU.A.Chl 445 17497 P.mU.A.A.G.fU.fC.A.A.fU.G.fU.A.fC*A*G*fC*fU*G*C 446 18458 17498 A.A.mC.mU.A.mU.mU.G.mC.mU.mU.mC.A.Chl 447 17500 P.mU.G.A.A.G.fC.A.A.fU.A.G.fU.fU*G*G*fU*G*fU*C 448 18459 17499 mC.A.A.mC.mU.A.mU.mU.G.mC.mU.mU.mC.A.Chl 449 17500 P.mU.G.A.A.G.fC.A.A.fU.A.G.fU.fU*G*G*fU*G*fU*C 450 18460 17501 G.mC.A.mU.A.mU.A.mU.A.mU.G.mU.A.Chl 451 17502 P.mU.A.fC.A.fU.A.fU.A.fU.A.fU.G.fC*fU*G*fU*G*fU*G 452 18461 17503 mU.G.mU.A.mC.A.mU.mU.G.A.mC.mU.A.Chl 453 17505 P.mU.A.G.fU.fC.A.A.fU.G.fU.A.fC.A*G*fC*fU*G*fC*C 454 18462 17504 mC.mU.G.mU.A.mC.A.mU.mU.G.A.mC.mU.A.Chl 455 17505 P.mU.A.G.fU.fC.A.A.fU.G.fU.A.fC.A*G*fC*fU*G*fC*C 456 18463 17506 A.G.mC.A.mU.A.mU.A.mU.A.mU.G.A.Chl 457 17507 P.mU.fC.A.fU.A.fU.A.fU.A.fU.G.fC.fU*G*fU*G*fU*G*U 458 18464 17508 mC.A.G.mC.A.A.mC.A.A.mU.mU.mC.A.Chl 459 17509 P.mU.G.A.A.fU.fU.G.fU.fU.G.fC.fU.G*fU*A*fU*fU*fU*C 460 18465 17510 mC.A.mU.A.mU.A.mU.A.mU.G.mU.mU.A.Chl 461 17511 P.mU.A.A.fC.A.fU.A.fU.A.fU.A.fU.G*fC*fU*G*fU*G*U 462 18466 17512 mU.mU.G.mC.mU.mU.mC.A.G.mC.mU.mC.A.Chl 463 17514 P.mU.G.A.G.fC.fU.G.A.A.G.fC.A.A*fU*A*G*fU*fU*G 464 18467 17513 A.mU.mU.G.mC.mU.mU.mC.A.G.mC.mU.mC.A.Chl 465 17514 P.mU.G.A.G.fC.fU.G.A.A.G.fC.A.A*fU*A*G*fU*fU*G 466 18468 17515 A.mC.A.G.mC.A.mU.A.mU.A.mU.A.A.Chl 467 17516 P.mU.fU.A.fU.A.fU.A.fU.G.fC.fU.G.fU*G*fU*G*fU*A*C 468 18469 17517 A.mU.mU.G.mC.mU.mU.mC.A.G.mC.mU.A.Chl 469 17519 P.mU.A.G.fC.fU.G.A.A.G.fC.A.A.fU*A*G*fU*fU*G*G 470 18470 17518 mU.A.mU.mU.G.mC.mU.mU.mC.A.G.mC.mU.A.Chl 471 17519 P.mU.A.G.fC.fU.G.A.A.G.fC.A.A.fU*A*G*fU*fU*G*G 472 18471 17520 mC.A.G.A.G.mU.A.mC.A.mC.A.mC.A.Chl 473 17521 P.mU.G.fU.G.fU.G.fU.A.fC.fU.fC.fU.G*C*fU*fU*G*A*A 474 18472 17522 mU.mC.A.A.G.mC.A.G.A.G.mU.A.A.Chl 475 17523 P.mU.fU.A.fC.fU.fC.fU.G.fC.fU.fU.G.A*A*fC*fU*fU*G*U 476 18473 17524 A.G.mC.A.G.A.G.mU.A.mC.A.mC.A.Chl 477 17525 P.mU.G.fU.G.fU.A.fC.fU.fC.fU.G.fC.fU*fU*G*A*A*fC*U 478 18474 17526 G.A.mC.A.A.G.mU.mU.mC.A.A.G.A.Chl 479 17527 P.mU.fC.fU.fU.G.A.A.fC.fU.fU.G.fU.fC*A*fU*A*G*A*U 480 18475 17528 mC.mU.A.mU.G.A.mC.A.A.G.mU.mU.A.Chl 481 17529 P.mU.A.A.fC.fU.fU.G.fU.fC.A.fU.A.G*A*fU*fU*fU*fC*G 482 18476 17530 G.mC.A.G.A.G.mU.A.mC.A.mC.A.A.Chl 483 17531 P.mU.fU.G.fU.G.fU.A.fC.fU.fC.fU.G.fC*fU*fU*G*A*A*C 484 18477 17532 mU.G.A.mC.A.A.G.mU.mU.mCA.A.A.Chl 485 17533 P.mU.fU.fU.G.A.A.fC.fU.fU.GfU.fC.A*fU*A*G*A*fU*U 486 18478 17534 mU.A.mC.A.mC.A.mC.A.G.mCA.mU.A.Chl 487 17535 P.mU.A.fU.G.fC.fU.G.fU.G.fUG.fU.A*fC*fU*fC*fU*G*C 488 18479 17536 A.A.mC.G.A.A.A.mU.mC.mUA.mU.A.Chl 489 17537 P.mU.A.fU.A.G.A.fU.fU.fU.fCG.fU.fU*G*fU*G*G*G*U 490 18480 17538 mU.mU.G.A.mC.mU.mU.mC.mC.GmC.A.A.Chl 491 17539 P.mU.fU.G.fC.G.G.A.A.G.fUfC.A.A*fU*G*fU*A*fC*A 492 18481 17540 A.mC.A.A.mC.G.A.A.A.mUmC.mU.A.Chl 493 17541 P.mU.A.G.A.fU.fU.fU.fC.G.fUfU.G.fU*G*G*G*fU*fU*U 494 18482 17542 mU.mC.A.A.mC.A.mC.A.mU.mCA.G.A.Chl 495 17543 P.mU.fC.fU.G.A.fU.G.fU.G.fUfU.G.A*A*G*A*A*fC*A 496 18483 17544 A.mC.A.A.G.mU.mU.mC.A.AG.mC.A.Chl 497 17545 P.mU.G.fC.fU.fU.G.A.A.fC.fUfU.G.fU*fC*A*fU*A*G*A 498 18484 17546 A.mU.mC.mU.A.mU.G.A.mC.AA.G.A.Chl 499 17547 P.mU.fC.fU.fU.G.fU.fC.A.fU.AG.A.fU*fU*fU*fC*G*fU*U 500 Rat Targeting TGFB1 18715 18691 G.A.A.A.mU.A.mU.A.G.mC.A.A.A-chol 503 18692 P.mU.fU.fU.G.fC.fU.A.fU.A.fU.fU.fU.fC*fU*G*G*fU*A 504 *G 18716 18693 G.A.A.mC.mU.mC.mU.A.mC.mC.A.G.A-chol 505 18694 P.mU.fC.fU.G.G.fU.A.G.A.G.fU.fU.fC*fU*A*fC*G*fU*G 506 18717 18695 G.mC.A.A.A.G.A.mU.A.A.mU.G.A-chol 507 18696 P.mU.fC.A.fU.fU.A.fU.fC.fU.fU.fU.G.fC*fU*G*fU*fC*A 508 *C 18718 18697 A.A.mC.mU.mC.mU.A.mC.mC.A.G.A.A-chol 509 18698 P.mU.fU.fC.fU.G.G.fU.A.G.A.G.fU.fU*fC*fU*A*fC*G*U 510 18719 18699 A.mC.mU.mC.mU.A.mC.mC.A.G.A.A.A-chol 511 18700 P.mU.fU.fU.fC.fU.G.G.fU.A.G.A.G.fU*fU*fC*fU*A*fC*G 512 18720 18701 A.mC.A.G.mC.A.A.A.G.A.mU.A.A-chol 513 18702 P.mU.fU.A.fU.fC.fU.fU.fU.G.fC.fU.G.fU*fC*A*fC*A*A*G 514 18721 18703 mC.A.A.mU.mC.mU.A.mU.G.A.mC.A.A-chol 515 18704 P.mU.fU.G.fU.fC.A.fU.A.G.A.fU.fU.G*fC*G*fU*fU*G*U 516 18722 18705 A.G.A.mU.mU.mC.A.A.G.mU.mC.A.A-chol 517 18706 P.mU.fU.G.A.fC.fU.fU.G.A.A.fU.fC.fU*fC*fU*G*fC*A*G 518 18723 18707 mC.mU.G.mU.G.G.A.G.mC.A.A.mC.A-chol 519 18708 P.mU.G.fU.fU.G.fC.fU.fC.fC.A.fC.A.G*fU*fU*G*A*fC*U 520 18724 18709 mU.G.A.mC.A.G.mC.A.A.A.G.A.A-chol 521 18710 P.mU.fU.fC.fU.fU.fU.G.fC.fU.G.fU.fC.A*fC*A*A*G*A*G 522 18725 18711 A.mU.G.A.mC.A.A.A.A.mC.mC.A.A-chol 523 18712 P.mU.fU.G.G.fU.fU.fU.fU.G.fU.fC.A.fU*A*G*A*fU*fU*G 524 18726 18713 G.A.G.A.mU.mU.mC.A.A.G.mU.mC.A-chol 525 18714 P.mU.G.A.fC.fU.fU.G.A.A.fU.fC.fU.fC*fU*G*fC*A*G*G 526

TABLE 5 Inhibition of gene expression with hTGFB1 ori sequences % Expression Target Gene Gene SEQ ID hTGFB1 0.025 nM HeLa Duplex ID Region Ref Pos NO Sense Sequence cells 15732 CDS 954 527 CGCGGGACUAUCCACCUGCAAGACA 57.3% 15733 CDS 956 528 CGGGACUAUCCACCUGCAAGACUAA 38.2% 15734 CDS 957 529 GGGACUAUCCACCUGCAAGACUAUA 49.1% 15735 CDS 961 530 CUAUCCACCUGCAAGACUAUCGACA 34.9% 15736 CDS 962 531 UAUCCACCUGCAAGACUAUCGACAA 39.4% 15737 CDS 964 532 UCCACCUGCAAGACUAUCGACAUGA 44.4% 15738 CDS 965 533 CCACCUGCAAGACUAUCGACAUGGA 53.3% 15739 CDS 966 534 CACCUGCAAGACUAUCGACAUGGAA 52.8% 15740 CDS 967 535 ACCUGCAAGACUAUCGACAUGGAGA 46.2% 15741 CDS 968 536 CCUGCAAGACUAUCGACAUGGAGCA 48.1% 15742 CDS 1209 537 AAUGGUGGAAACCCACAACGAAAUA 36.7% 15743 CDS 1210 538 AUGGUGGAAACCCACAACGAAAUCA 28.8% 15744 CDS 1211 539 UGGUGGAAACCCACAACGAAAUCUA 23.1% 15745 CDS 1212 540 GGUGGAAACCCACAACGAAAUCUAA 13.2% 15746 CDS 1213 541 GUGGAAACCCACAACGAAAUCUAUA 21.1% 15747 CDS 1214 542 UGGAAACCCACAACGAAAUCUAUGA 28.7% 15748 CDS 1215 543 GGAAACCCACAACGAAAUCUAUGAA 32.9% 15749 CDS 1216 544 GAAACCCACAACGAAAUCUAUGACA 41.5% 15750 CDS 1217 545 AAACCCACAACGAAAUCUAUGACAA 29.9% 15751 CDS 1218 546 AACCCACAACGAAAUCUAUGACAAA 16.4% 15752 CDS 1219 547 ACCCACAACGAAAUCUAUGACAAGA 23.3% 15753 CDS 1220 548 CCCACAACGAAAUCUAUGACAAGUA 37.5% 15754 CDS 1221 549 CCACAACGAAAUCUAUGACAAGUUA 19.1% 15755 CDS 1222 550 CACAACGAAAUCUAUGACAAGUUCA 14.4% 15756 CDS 1224 551 CAACGAAAUCUAUGACAAGUUCAAA 20.1% 15757 CDS 1225 552 AACGAAAUCUAUGACAAGUUCAAGA 18.3% 15758 CDS 1226 553 ACGAAAUCUAUGACAAGUUCAAGCA 23.2% 15759 CDS 1227 554 CGAAAUCUAUGACAAGUUCAAGCAA 29.0% 15760 CDS 1228 555 GAAAUCUAUGACAAGUUCAAGCAGA 15.6% 15761 CDS 1229 556 AAAUCUAUGACAAGUUCAAGCAGAA 32.3% 15762 CDS 1230 557 AAUCUAUGACAAGUUCAAGCAGAGA 36.1% 15763 CDS 1231 558 AUCUAUGACAAGUUCAAGCAGAGUA 30.6% 15764 CDS 1232 559 UCUAUGACAAGUUCAAGCAGAGUAA 24.9% 15765 CDS 1233 560 CUAUGACAAGUUCAAGCAGAGUACA 15.9% 15766 CDS 1234 561 UAUGACAAGUUCAAGCAGAGUACAA 31.2% 15767 CDS 1235 562 AUGACAAGUUCAAGCAGAGUACACA 17.2% 15768 CDS 1236 563 UGACAAGUUCAAGCAGAGUACACAA 23.5% 15769 CDS 1237 564 GACAAGUUCAAGCAGAGUACACACA 24.5% 15770 CDS 1238 565 ACAAGUUCAAGCAGAGUACACACAA 38.5% 15771 CDS 1240 566 AAGUUCAAGCAGAGUACACACAGCA 38.7% 15772 CDS 1241 567 AGUUCAAGCAGAGUACACACAGCAA 34.3% 15773 CDS 1242 568 GUUCAAGCAGAGUACACACAGCAUA 20.8% 15774 CDS 1243 569 UUCAAGCAGAGUACACACAGCAUAA 33.4% 15775 CDS 1244 570 UCAAGCAGAGUACACACAGCAUAUA 19.6% 15776 CDS 1245 571 CAAGCAGAGUACACACAGCAUAUAA 25.5% 15777 CDS 1246 572 AAGCAGAGUACACACAGCAUAUAUA 12.8% 15778 CDS 1247 573 AGCAGAGUACACACAGCAUAUAUAA 27.6% 15779 CDS 1248 574 GCAGAGUACACACAGCAUAUAUAUA 15.9% 15780 CDS 1249 575 CAGAGUACACACAGCAUAUAUAUGA 24.1% 15781 CDS 1250 576 AGAGUACACACAGCAUAUAUAUGUA 22.6% 15782 CDS 1251 577 GAGUACACACAGCAUAUAUAUGUUA 26.7% 15783 CDS 1252 578 AGUACACACAGCAUAUAUAUGUUCA 66.6% 15784 CDS 1254 579 UACACACAGCAUAUAUAUGUUCUUA 33.6% 15785 CDS 1262 580 GCAUAUAUAUGUUCUUCAACACAUA 40.4% 15786 CDS 1263 581 CAUAUAUAUGUUCUUCAACACAUCA 42.5% 15787 CDS 1264 582 AUAUAUAUGUUCUUCAACACAUCAA 27.2% 15788 CDS 1265 583 UAUAUAUGUUCUUCAACACAUCAGA 23.2% 15789 CDS 1266 584 AUAUAUGUUCUUCAACACAUCAGAA 35.5% 15790 CDS 1267 585 UAUAUGUUCUUCAACACAUCAGAGA 34.6% 15791 CDS 1268 586 AUAUGUUCUUCAACACAUCAGAGCA 29.7% 15792 CDS 1269 587 UAUGUUCUUCAACACAUCAGAGCUA 35.4% 15793 CDS 1270 588 AUGUUCUUCAACACAUCAGAGCUCA 35.2% 15794 CDS 1335 589 GCUGCGUCUGCUGAGGCUCAAGUUA 28.0% 15795 CDS 1336 590 CUGCGUCUGCUGAGGCUCAAGUUAA 32.1% 15796 CDS 1337 591 UGCGUCUGCUGAGGCUCAAGUUAAA 25.5% 15797 CDS 1338 592 GCGUCUGCUGAGGCUCAAGUUAAAA 59.7% 15798 CDS 1339 593 CGUCUGCUGAGGCUCAAGUUAAAAA 52.8% 15799 CDS 1340 594 GUCUGCUGAGGCUCAAGUUAAAAGA 47.9% 15800 CDS 1341 595 UCUGCUGAGGCUCAAGUUAAAAGUA 49.8% 15801 CDS 1342 596 CUGCUGAGGCUCAAGUUAAAAGUGA 50.7% 15802 CDS 1343 597 UGCUGAGGCUCAAGUUAAAAGUGGA 43.4% 15803 CDS 1344 598 GCUGAGGCUCAAGUUAAAAGUGGAA 52.6% 15804 CDS 1345 599 CUGAGGCUCAAGUUAAAAGUGGAGA 73.3% 15805 CDS 1346 600 UGAGGCUCAAGUUAAAAGUGGAGCA 58.0% 15806 CDS 1347 601 GAGGCUCAAGUUAAAAGUGGAGCAA 64.9% 15807 CDS 1348 602 AGGCUCAAGUUAAAAGUGGAGCAGA 68.1% 15808 CDS 1349 603 GGCUCAAGUUAAAAGUGGAGCAGCA 73.8% 15809 CDS 1350 604 GCUCAAGUUAAAAGUGGAGCAGCAA 78.8% 15810 CDS 1351 605 CUCAAGUUAAAAGUGGAGCAGCACA 76.6% 15811 CDS 1352 606 UCAAGUUAAAAGUGGAGCAGCACGA 72.9% 15812 CDS 1369 607 CAGCACGUGGAGCUGUACCAGAAAA 69.8% 15813 CDS 1370 608 AGCACGUGGAGCUGUACCAGAAAUA 69.7% 15814 CDS 1371 609 GCACGUGGAGCUGUACCAGAAAUAA 73.3% 15815 CDS 1372 610 CACGUGGAGCUGUACCAGAAAUACA 55.0% 15816 CDS 1373 611 ACGUGGAGCUGUACCAGAAAUACAA 63.8% 15817 CDS 1374 612 CGUGGAGCUGUACCAGAAAUACAGA 85.7% 15818 CDS 1375 613 GUGGAGCUGUACCAGAAAUACAGCA 85.0% 15819 CDS 1376 614 UGGAGCUGUACCAGAAAUACAGCAA 82.5% 15820 CDS 1377 615 GGAGCUGUACCAGAAAUACAGCAAA 43.1% 15821 CDS 1378 616 GAGCUGUACCAGAAAUACAGCAACA 58.5% 15822 CDS 1379 617 AGCUGUACCAGAAAUACAGCAACAA 48.1% 15823 CDS 1380 618 GCUGUACCAGAAAUACAGCAACAAA 48.1% 15824 CDS 1381 619 CUGUACCAGAAAUACAGCAACAAUA 35.0% 15825 CDS 1382 620 UGUACCAGAAAUACAGCAACAAUUA 36.4% 15826 CDS 1383 621 GUACCAGAAAUACAGCAACAAUUCA 24.6% 15827 CDS 1384 622 UACCAGAAAUACAGCAACAAUUCCA 33.4% 15828 CDS 1385 623 ACCAGAAAUACAGCAACAAUUCCUA 121.5% 15829 CDS 1386 624 CCAGAAAUACAGCAACAAUUCCUGA 62.1% 15830 CDS 1387 625 CAGAAAUACAGCAACAAUUCCUGGA 98.3% 15831 CDS 1390 626 AAAUACAGCAACAAUUCCUGGCGAA 36.6% 15832 CDS 1391 627 AAUACAGCAACAAUUCCUGGCGAUA 39.5% 15833 CDS 1392 628 AUACAGCAACAAUUCCUGGCGAUAA 40.0% 15834 CDS 1393 629 UACAGCAACAAUUCCUGGCGAUACA 89.4% 15835 CDS 1394 630 ACAGCAACAAUUCCUGGCGAUACCA 62.3% 15836 CDS 1396 631 AGCAACAAUUCCUGGCGAUACCUCA 41.0% 15837 CDS 1441 632 AGCGACUCGCCAGAGUGGUUAUCUA 31.2% 15838 CDS 1442 633 GCGACUCGCCAGAGUGGUUAUCUUA 46.2% 15839 CDS 1443 634 CGACUCGCCAGAGUGGUUAUCUUUA 46.8% 15840 CDS 1444 635 GACUCGCCAGAGUGGUUAUCUUUUA 50.6% 15841 CDS 1445 636 ACUCGCCAGAGUGGUUAUCUUUUGA 50.8% 15842 CDS 1446 637 CUCGCCAGAGUGGUUAUCUUUUGAA 71.8% 15843 CDS 1447 638 UCGCCAGAGUGGUUAUCUUUUGAUA 43.7% 15844 CDS 1448 639 CGCCAGAGUGGUUAUCUUUUGAUGA 42.1% 15845 CDS 1449 640 GCCAGAGUGGUUAUCUUUUGAUGUA 31.0% 15846 CDS 1450 641 CCAGAGUGGUUAUCUUUUGAUGUCA 46.0% 15847 CDS 1451 642 CAGAGUGGUUAUCUUUUGAUGUCAA 40.2% 15848 CDS 1452 643 AGAGUGGUUAUCUUUUGAUGUCACA 38.5% 15849 CDS 1453 644 GAGUGGUUAUCUUUUGAUGUCACCA 67.4% 15850 CDS 1454 645 AGUGGUUAUCUUUUGAUGUCACCGA 57.4% 15851 CDS 1455 646 GUGGUUAUCUUUUGAUGUCACCGGA 40.6% 15852 CDS 1456 647 UGGUUAUCUUUUGAUGUCACCGGAA 70.5% 15853 CDS 1457 648 GGUUAUCUUUUGAUGUCACCGGAGA 82.8% 15854 CDS 1458 649 GUUAUCUUUUGAUGUCACCGGAGUA 74.8% 15855 CDS 1459 650 UUAUCUUUUGAUGUCACCGGAGUUA 86.8% 15856 CDS 1460 651 UAUCUUUUGAUGUCACCGGAGUUGA 76.5% 15857 CDS 1551 652 CAGCAGGGAUAACACACUGCAAGUA 70.5% 15858 CDS 1552 653 AGCAGGGAUAACACACUGCAAGUGA 60.5% 15859 CDS 1553 654 GCAGGGAUAACACACUGCAAGUGGA 43.5% 15860 CDS 1554 655 CAGGGAUAACACACUGCAAGUGGAA 56.3% 15861 CDS 1555 656 AGGGAUAACACACUGCAAGUGGACA 63.9% 15862 CDS 1556 657 GGGAUAACACACUGCAAGUGGACAA 66.9% 15863 CDS 1558 658 GAUAACACACUGCAAGUGGACAUCA 62.2% 15864 CDS 1559 659 AUAACACACUGCAAGUGGACAUCAA 40.5% 15865 CDS 1560 660 UAACACACUGCAAGUGGACAUCAAA 57.9% 15866 CDS 1610 661 ACCUGGCCACCAUUCAUGGCAUGAA 69.4% 15867 CDS 1611 662 CCUGGCCACCAUUCAUGGCAUGAAA 49.1% 15868 CDS 1612 663 CUGGCCACCAUUCAUGGCAUGAACA 31.9% 15869 CDS 1705 664 CGAGCCCUGGACACCAACUAUUGCA 56.4% 15870 CDS 1706 665 GAGCCCUGGACACCAACUAUUGCUA 42.6% 15871 CDS 1707 666 AGCCCUGGACACCAACUAUUGCUUA 29.8% 15872 CDS 1708 667 GCCCUGGACACCAACUAUUGCUUCA 19.8% 15873 CDS 1709 668 CCCUGGACACCAACUAUUGCUUCAA 37.7% 15874 CDS 1710 669 CCUGGACACCAACUAUUGCUUCAGA 44.0% 15875 CDS 1711 670 CUGGACACCAACUAUUGCUUCAGCA 35.8% 15876 CDS 1712 671 UGGACACCAACUAUUGCUUCAGCUA 31.5% 15877 CDS 1713 672 GGACACCAACUAUUGCUUCAGCUCA 27.3% 15878 CDS 1714 673 GACACCAACUAUUGCUUCAGCUCCA 44.7% 15879 CDS 1715 674 ACACCAACUAUUGCUUCAGCUCCAA 44.9% 15880 CDS 1754 675 GCGUGCGGCAGCUGUACAUUGACUA 23.9% 15881 CDS 1755 676 CGUGCGGCAGCUGUACAUUGACUUA 18.3% 15882 CDS 1756 677 GUGCGGCAGCUGUACAUUGACUUCA 41.2% 15883 CDS 1757 678 UGCGGCAGCUGUACAUUGACUUCCA 26.4% 15884 CDS 1759 679 CGGCAGCUGUACAUUGACUUCCGCA 28.0% 15885 CDS 1760 680 GGCAGCUGUACAUUGACUUCCGCAA 22.8% 15886 CDS 1761 681 GCAGCUGUACAUUGACUUCCGCAAA 34.1% 15887 CDS 1762 682 CAGCUGUACAUUGACUUCCGCAAGA 36.3% 15888 CDS 1763 683 AGCUGUACAUUGACUUCCGCAAGGA 84.1% 15889 CDS 1849 684 UGCCCCUACAUUUGGAGCCUGGACA 93.0% 15890 CDS 1889 685 UCCUGGCCCUGUACAACCAGCAUAA 51.7% 15891 CDS 1890 686 CCUGGCCCUGUACAACCAGCAUAAA 71.9% 15892 CDS 1891 687 CUGGCCCUGUACAACCAGCAUAACA 36.1% 15893 CDS 1997 688 AGGUGGAGCAGCUGUCCAACAUGAA 60.9% 15894 3UTR 2115 689 CAUGGGGGCUGUAUUUAAGGACACA 57.2% 15895 3UTR 2155 690 CCUGGGGCCCCAUUAAAGAUGGAGA 86.0% 15896 3UTR 2156 691 CUGGGGCCCCAUUAAAGAUGGAGAA 73.3% 15897 3UTR 2157 692 UGGGGCCCCAUUAAAGAUGGAGAGA 68.8% 15898 3UTR 2158 693 GGGGCCCCAUUAAAGAUGGAGAGAA 65.8% 15899 3UTR 2159 694 GGGCCCCAUUAAAGAUGGAGAGAGA 42.7% 15900 3UTR 2160 695 GGCCCCAUUAAAGAUGGAGAGAGGA 34.4% 15901 3UTR 2161 696 GCCCCAUUAAAGAUGGAGAGAGGAA 56.0% 15902 3UTR 2162 697 CCCCAUUAAAGAUGGAGAGAGGACA 74.9% 15903 3UTR 2163 698 CCCAUUAAAGAUGGAGAGAGGACUA 79.6% 15904 3UTR 2180 699 GAGGACUGCGGAUCUCUGUGUCAUA 98.3% 15905 3UTR 2275 700 CUCCUGCCUGUCUGCACUAUUCCUA 100.2% 15906 3UTR 2276 701 UCCUGCCUGUCUGCACUAUUCCUUA 103.8% 15907 3UTR 2277 702 CCUGCCUGUCUGCACUAUUCCUUUA 110.4% 15908 3UTR 2278 703 CUGCCUGUCUGCACUAUUCCUUUGA 105.2% 15909 3UTR 2279 704 UGCCUGUCUGCACUAUUCCUUUGCA 118.8% 15910 3UTR 2325 705 CAGUGGGGAACACUACUGUAGUUAA 112.2% 15911 3UTR 2326 706 AGUGGGGAACACUACUGUAGUUAGA 107.7% 15912 3UTR 2327 707 GUGGGGAACACUACUGUAGUUAGAA 108.6% 15913 3UTR 2328 708 UGGGGAACACUACUGUAGUUAGAUA N/A

TABLE 6 Inhibition of gene expression with hTGFB2 ori sequences % Oligo Gene Expression 25-mer Sense Strand (position 25 of SS, id Region Ref Pos A549 0.1 nM replaced with A) SEQ ID NO 15451 5UTR/CDS 651   98% UUUUAAAAAAUGCACUACUGUGUGC 709 15452 CDS 654 102.2%  UAAAAAAUGCACUACUGUGUGCUGA 710 15453 CDS 730 83.7% GCAGCACACUCGAUAUGGACCAGUU 711 15454 CDS 732 80.3% AGCACACUCGAUAUGGACCAGUUCA 712 15455 CDS 733 79.6% GCACACUCGAUAUGGACCAGUUCAU 713 15456 CDS 734 89.1% CACACUCGAUAUGGACCAGUUCAUG 714 15457 CDS 735 87.8% ACACUCGAUAUGGACCAGUUCAUGC 715 15458 CDS 736 95.3% CACUCGAUAUGGACCAGUUCAUGCG 716 15459 CDS 847 103.8%  UCCCCCCGGAGGUGAUUUCCAUCUA 717 15460 CDS 848 83.6% CCCCCCGGAGGUGAUUUCCAUCUAC 718 15461 CDS 851 72.2% CCCGGAGGUGAUUUCCAUCUACAAC 719 15462 CDS 853 85.8% CGGAGGUGAUUUCCAUCUACAACAG 720 15463 CDS 855 67.1% GAGGUGAUUUCCAUCUACAACAGCA 721 15464 CDS 952 68.9% ACUACGCCAAGGAGGUUUACAAAAU 722 15465 CDS 963 81.1% GAGGUUUACAAAAUAGACAUGCCGC 723 15466 CDS 1107 82.1% UUCUACAGACCCUACUUCAGAAUUG 724 15467 CDS 1108 99.1% UCUACAGACCCUACUUCAGAAUUGU 725 15468 CDS 1109 95.1% CUACAGACCCUACUUCAGAAUUGUU 726 15469 CDS 1129 90.4% UUGUUCGAUUUGACGUCUCAGCAAU 727 15470 CDS 1130 76.7% UGUUCGAUUUGACGUCUCAGCAAUG 728 15471 CDS 1131 79.7% GUUCGAUUUGACGUCUCAGCAAUGG 729 15472 CDS 1132 87.5% UUCGAUUUGACGUCUCAGCAAUGGA 730 15473 CDS 1144 66.9% UCUCAGCAAUGGAGAAGAAUGCUUC 731 15474 CDS 1145 76.6% CUCAGCAAUGGAGAAGAAUGCUUCC 732 15475 CDS 1147 88.9% CAGCAAUGGAGAAGAAUGCUUCCAA 733 15476 CDS 1162 84.5% AUGCUUCCAAUUUGGUGAAAGCAGA 734 15477 CDS 1163 89.2% UGCUUCCAAUUUGGUGAAAGCAGAG 735 15478 CDS 1165 86.6% CUUCCAAUUUGGUGAAAGCAGAGUU 736 15479 CDS 1177 61.2% UGAAAGCAGAGUUCAGAGUCUUUCG 737 15480 CDS 1185 92.6% GAGUUCAGAGUCUUUCGUUUGCAGA 738 15481 CDS 1219 99.6% CCAGAGUGCCUGAACAACGGAUUGA 739 15482 CDS 1224 94.0% GUGCCUGAACAACGGAUUGAGCUAU 740 15483 CDS 1225 88.1% UGCCUGAACAACGGAUUGAGCUAUA 741 15484 CDS 1228 59.3% CUGAACAACGGAUUGAGCUAUAUCA 742 15485 CDS 1229 77.5% UGAACAACGGAUUGAGCUAUAUCAG 743 15486 CDS 1230 61.5% GAACAACGGAUUGAGCUAUAUCAGA 744 15487 CDS 1233 84.5% CAACGGAUUGAGCUAUAUCAGAUUC 745 15488 CDS 1238 87.7% GAUUGAGCUAUAUCAGAUUCUCAAG 746 15489 CDS 1239 78.7% AUUGAGCUAUAUCAGAUUCUCAAGU 747 15490 CDS 1240 94.1% UUGAGCUAUAUCAGAUUCUCAAGUC 748 15491 CDS 1247 92.6% AUAUCAGAUUCUCAAGUCCAAAGAU 749 15492 CDS 1256 94.3% UCUCAAGUCCAAAGAUUUAACAUCU 750 15493 CDS 1259 99.1% CAAGUCCAAAGAUUUAACAUCUCCA 751 15494 CDS 1286 87.4% CCAGCGCUACAUCGACAGCAAAGUU 752 15495 CDS 1288 84.5% AGCGCUACAUCGACAGCAAAGUUGU 753 15496 CDS 1289 60.1% GCGCUACAUCGACAGCAAAGUUGUG 754 15497 CDS 1292 78.8% CUACAUCGACAGCAAAGUUGUGAAA 755 15498 CDS 1331 80.1% CGAAUGGCUCUCCUUCGAUGUAACU 756 15499 CDS 1353 62.4% ACUGAUGCUGUUCAUGAAUGGCUUC 757 15500 CDS 1361 74.3% UGUUCAUGAAUGGCUUCACCAUAAA 758 15501 CDS 1362 75.1% GUUCAUGAAUGGCUUCACCAUAAAG 759 15502 CDS 1363 87.2% UUCAUGAAUGGCUUCACCAUAAAGA 760 15503 CDS 1364 70.4% UCAUGAAUGGCUUCACCAUAAAGAC 761 15504 CDS 1365 100.7%  CAUGAAUGGCUUCACCAUAAAGACA 762 15505 CDS 1368 100.1%  GAAUGGCUUCACCAUAAAGACAGGA 763 15506 CDS 1398 92.0% GGAUUUAAAAUAAGCUUACACUGUC 764 15507 CDS 1399 83.2% GAUUUAAAAUAAGCUUACACUGUCC 765 15508 CDS 1415 85.6% ACACUGUCCCUGCUGCACUUUUGUA 766 15509 CDS 1418 97.4% CUGUCCCUGCUGCACUUUUGUACCA 767 15510 CDS 1420 59.1% GUCCCUGCUGCACUUUUGUACCAUC 768 15511 CDS 1421 73.7% UCCCUGCUGCACUUUUGUACCAUCU 769 15512 CDS 1422 79.5% CCCUGCUGCACUUUUGUACCAUCUA 770 15513 CDS 1451 62.7% UUACAUCAUCCCAAAUAAAAGUGAA 771 15514 CDS 1452 76.0% UACAUCAUCCCAAAUAAAAGUGAAG 772 15515 CDS 1470 44.7% AGUGAAGAACUAGAAGCAAGAUUUG 773 15516 CDS 1472 75.6% UGAAGAACUAGAAGCAAGAUUUGCA 774 15517 CDS 1474 96.8% AAGAACUAGAAGCAAGAUUUGCAGG 775 15518 CDS 1475 94.3% AGAACUAGAAGCAAGAUUUGCAGGU 776 15519 CDS 1476 63.3% GAACUAGAAGCAAGAUUUGCAGGUA 777 15520 CDS 1480 65.9% UAGAAGCAAGAUUUGCAGGUAUUGA 778 15521 CDS 1481 59.6% AGAAGCAAGAUUUGCAGGUAUUGAU 779 15522 CDS 1482 56.0% GAAGCAAGAUUUGCAGGUAUUGAUG 780 15523 CDS 1483 69.2% AAGCAAGAUUUGCAGGUAUUGAUGG 781 15524 CDS 1484 64.5% AGCAAGAUUUGCAGGUAUUGAUGGC 782 15525 CDS 1485 92.0% GCAAGAUUUGCAGGUAUUGAUGGCA 783 15526 CDS 1486 101.7%  CAAGAUUUGCAGGUAUUGAUGGCAC 784 15527 CDS 1496 103.3%  AGGUAUUGAUGGCACCUCCACAUAU 785 15528 CDS 1503 102.3%  GAUGGCACCUCCACAUAUACCAGUG 786 15529 CDS 1506 86.6% GGCACCUCCACAUAUACCAGUGGUG 787 15530 CDS 1510 79.9% CCUCCACAUAUACCAGUGGUGAUCA 788 15531 CDS 1511 44.9% CUCCACAUAUACCAGUGGUGAUCAG 789 15532 CDS 1512 57.3% UCCACAUAUACCAGUGGUGAUCAGA 790 15533 CDS 1517 64.9% AUAUACCAGUGGUGAUCAGAAAACU 791 15534 CDS 1518 90.8% UAUACCAGUGGUGAUCAGAAAACUA 792 15535 CDS 1520 47.1% UACCAGUGGUGAUCAGAAAACUAUA 793 15536 CDS 1526 55.7% UGGUGAUCAGAAAACUAUAAAGUCC 794 15537 CDS 1527 89.6% GGUGAUCAGAAAACUAUAAAGUCCA 795 15538 CDS 1529 92.4% UGAUCAGAAAACUAUAAAGUCCACU 796 15539 CDS 1531 87.2% AUCAGAAAACUAUAAAGUCCACUAG 797 15540 CDS 1532 93.4% UCAGAAAACUAUAAAGUCCACUAGG 798 15541 CDS 1575 78.4% ACCCCACAUCUCCUGCUAAUGUUAU 799 15542 CDS 1576 84.6% CCCCACAUCUCCUGCUAAUGUUAUU 800 15543 CDS 1579 95.9% CACAUCUCCUGCUAAUGUUAUUGCC 801 15544 CDS 1591 89.6% UAAUGUUAUUGCCCUCCUACAGACU 802 15545 CDS 1592 85.0% AAUGUUAUUGCCCUCCUACAGACUU 803 15546 CDS 1598 51.2% AUUGCCCUCCUACAGACUUGAGUCA 804 15547 CDS 1650 39.4% GCUUUGGAUGCGGCCUAUUGCUUUA 805 15548 CDS 1652 82.3% UUUGGAUGCGGCCUAUUGCUUUAGA 806 15549 CDS 1653 86.1% UUGGAUGCGGCCUAUUGCUUUAGAA 807 15550 CDS 1655 80.0% GGAUGCGGCCUAUUGCUUUAGAAAU 808 15551 CDS 1657 72.3% AUGCGGCCUAUUGCUUUAGAAAUGU 809 15552 CDS 1658 72.2% UGCGGCCUAUUGCUUUAGAAAUGUG 810 15553 CDS 1659 57.8% GCGGCCUAUUGCUUUAGAAAUGUGC 811 15554 CDS 1660 83.4% CGGCCUAUUGCUUUAGAAAUGUGCA 812 15555 CDS 1662 79.3% GCCUAUUGCUUUAGAAAUGUGCAGG 813 15556 CDS 1663 86.3% CCUAUUGCUUUAGAAAUGUGCAGGA 814 15557 CDS 1664 84.8% CUAUUGCUUUAGAAAUGUGCAGGAU 815 15558 CDS 1665 71.1% UAUUGCUUUAGAAAUGUGCAGGAUA 816 15559 CDS 1666 61.8% AUUGCUUUAGAAAUGUGCAGGAUAA 817 15560 CDS 1667 84.9% UUGCUUUAGAAAUGUGCAGGAUAAU 818 15561 CDS 1668 82.8% UGCUUUAGAAAUGUGCAGGAUAAUU 819 15562 CDS 1670 69.8% CUUUAGAAAUGUGCAGGAUAAUUGC 820 15563 CDS 1671 90.2% UUUAGAAAUGUGCAGGAUAAUUGCU 821 15564 CDS 1672 68.6% UUAGAAAUGUGCAGGAUAAUUGCUG 822 15565 CDS 1678 74.2% AUGUGCAGGAUAAUUGCUGCCUACG 823 15566 CDS 1761 58.6% GGGUACAAUGCCAACUUCUGUGCUG 824 15567 CDS 1767 86.3% AAUGCCAACUUCUGUGCUGGAGCAU 825 15568 CDS 1782 83.7% GCUGGAGCAUGCCCGUAUUUAUGGA 826 15569 CDS 1783 86.9% CUGGAGCAUGCCCGUAUUUAUGGAG 827 15570 CDS 1786 90.5% GAGCAUGCCCGUAUUUAUGGAGUUC 828 15571 CDS 1787 91.1% AGCAUGCCCGUAUUUAUGGAGUUCA 829 15572 CDS 1788 68.0% GCAUGCCCGUAUUUAUGGAGUUCAG 830 15573 CDS 1789 75.7% CAUGCCCGUAUUUAUGGAGUUCAGA 831 15574 CDS 1796 88.9% GUAUUUAUGGAGUUCAGACACUCAG 832 15575 CDS 1800 52.5% UUAUGGAGUUCAGACACUCAGCACA 833 15576 CDS 1907 90.8% AACCAUUCUCUACUACAUUGGCAAA 834 15577 CDS 1924 70.2% UUGGCAAAACACCCAAGAUUGAACA 835 15578 CDS 1925 77.5% UGGCAAAACACCCAAGAUUGAACAG 836 15579 CDS/3UTR 1973 91.1% UUGCAAAUGCAGCUAAAAUUCUUGG 837 15580 3UTR 2020 70.1% CAAUGAUGAUGAUAAUGAUGAUGAC 838 15581 3UTR 2022 43.3% AUGAUGAUGAUAAUGAUGAUGACGA 839 15582 3UTR 2023 60.3% UGAUGAUGAUAAUGAUGAUGACGAC 840 15583 3UTR 2025 75.4% AUGAUGAUAAUGAUGAUGACGACGA 841 15584 3UTR 2026 40.8% UGAUGAUAAUGAUGAUGACGACGAC 842 15585 3UTR 2028 51.8% AUGAUAAUGAUGAUGACGACGACAA 843 15586 3UTR 2029 59.1% UGAUAAUGAUGAUGACGACGACAAC 844 15587 3UTR 2031 51.3% AUAAUGAUGAUGACGACGACAACGA 845 15588 3UTR 2032 32.7% UAAUGAUGAUGACGACGACAACGAU 846 15589 3UTR 2034 33.8% AUGAUGAUGACGACGACAACGAUGA 847 15590 3UTR 2035 57.0% UGAUGAUGACGACGACAACGAUGAU 848 15591 3UTR 2039 40.5% GAUGACGACGACAACGAUGAUGCUU 849 15592 3UTR 2045 56.8% GACGACAACGAUGAUGCUUGUAACA 850 15593 3UTR 2046 28.5% ACGACAACGAUGAUGCUUGUAACAA 851 15594 3UTR 2065 44.7% UAACAAGAAAACAUAAGAGAGCCUU 852 15595 3UTR 2066 58.3% AACAAGAAAACAUAAGAGAGCCUUG 853 15596 3UTR 2067 62.9% ACAAGAAAACAUAAGAGAGCCUUGG 854 15597 3UTR 2072 38.1% AAAACAUAAGAGAGCCUUGGUUCAU 855 15598 3UTR 2073 44.6% AAACAUAAGAGAGCCUUGGUUCAUC 856 15599 3UTR 2079 53.6% AAGAGAGCCUUGGUUCAUCAGUGUU 857 15600 3UTR 2081 33.2% GAGAGCCUUGGUUCAUCAGUGUUAA 858 15601 3UTR 2083 28.2% GAGCCUUGGUUCAUCAGUGUUAAAA 859 15602 3UTR 2110 46.5% UUUUUGAAAAGGCGGUACUAGUUCA 860 15603 3UTR 2116 56.1% AAAAGGCGGUACUAGUUCAGACACU 861 15604 3UTR 2117 60.9% AAAGGCGGUACUAGUUCAGACACUU 862 15605 3UTR 2136 76.8% ACACUUUGGAAGUUUGUGUUCUGUU 863 15606 3UTR 2137 29.5% CACUUUGGAAGUUUGUGUUCUGUUU 864 15607 3UTR 2140 62.6% UUUGGAAGUUUGUGUUCUGUUUGUU 865 15608 3UTR 2145 50.7% AAGUUUGUGUUCUGUUUGUUAAAAC 866 15609 3UTR 2147 62.9% GUUUGUGUUCUGUUUGUUAAAACUG 867 15610 3UTR 2148 59.7% UUUGUGUUCUGUUUGUUAAAACUGG 868 15611 3UTR 2149 50.3% UUGUGUUCUGUUUGUUAAAACUGGC 869 15612 3UTR 2150 49.8% UGUGUUCUGUUUGUUAAAACUGGCA 870 15613 3UTR 2152 55.2% UGUUCUGUUUGUUAAAACUGGCAUC 871 15614 3UTR 2153 82.2% GUUCUGUUUGUUAAAACUGGCAUCU 872 15615 3UTR 2154 70.0% UUCUGUUUGUUAAAACUGGCAUCUG 873 15616 3UTR 2155 45.5% UCUGUUUGUUAAAACUGGCAUCUGA 874 15617 3UTR 2156 54.9% CUGUUUGUUAAAACUGGCAUCUGAC 875 15618 3UTR 2189 40.4% AGUUGAAGGCCUUAUUCUACAUUUC 876 15619 3UTR 2190 34.1% GUUGAAGGCCUUAUUCUACAUUUCA 877 15620 3UTR 2207 91.3% ACAUUUCACCUACUUUGUAAGUGAG 878 15621 3UTR 2265 60.9% AAUAAACACUGGAAGAAUUUAUUAG 879 15622 3UTR 2267 36.4% UAAACACUGGAAGAAUUUAUUAGUG 880 15623 3UTR 2295 40.6% AUUAUGUGAACAACGACAACAACAA 881 15624 3UTR 2296 33.6% UUAUGUGAACAACGACAACAACAAC 882 15625 3UTR 2297 32.7% UAUGUGAACAACGACAACAACAACA 883 15626 3UTR 2298 40.8% AUGUGAACAACGACAACAACAACAA 884 15627 3UTR 2299 38.5% UGUGAACAACGACAACAACAACAAC 885 15628 3UTR 2301 84.2% UGAACAACGACAACAACAACAACAA 886 15629 3UTR 2302 43.2% GAACAACGACAACAACAACAACAAC 887 15630 3UTR 2304 57.8% ACAACGACAACAACAACAACAACAA 888 15631 3UTR 2305 44.3% CAACGACAACAACAACAACAACAAC 889 15632 3UTR 2308 38.7% CGACAACAACAACAACAACAACAAA 890 15633 3UTR 2309 37.4% GACAACAACAACAACAACAACAAAC 891 15634 3UTR 2314 73.5% CAACAACAACAACAACAAACAGGAA 892 15635 3UTR 2315 54.2% AACAACAACAACAACAAACAGGAAA 893 15636 3UTR 2389 30.7% CUUGAUUUUUCUGUAUUGCUAUGCA 894 15637 3UTR 2435 16.0% ACUCUUAGAGUUAACAGUGAGUUAU 895 15638 3UTR 2445 18.4% UUAACAGUGAGUUAUUUAUUGUGUG 896 15639 3UTR 2471 36.3% UACUAUAUAAUGAACGUUUCAUUGC 897 15640 3UTR 2472 73.3% ACUAUAUAAUGAACGUUUCAUUGCC 898 15641 3UTR 2484 63.4% ACGUUUCAUUGCCCUUGGAAAAUAA 899 15642 3UTR 2488 65.4% UUCAUUGCCCUUGGAAAAUAAAACA 900 15643 3UTR 2493 39.3% UGCCCUUGGAAAAUAAAACAGGUGU 901 15644 3UTR 2519 66.7% UAAAGUGGAGACCAAAUACUUUGCC 902 15645 3UTR 2520 40.1% AAAGUGGAGACCAAAUACUUUGCCA 903 15646 3UTR 2526 40.9% GAGACCAAAUACUUUGCCAGAAACU 904 15647 3UTR 2527 41.5% AGACCAAAUACUUUGCCAGAAACUC 905 15648 3UTR 2528 47.6% GACCAAAUACUUUGCCAGAAACUCA 906 15649 3UTR 2529 47.6% ACCAAAUACUUUGCCAGAAACUCAU 907 15650 3UTR 2530 31.9% CCAAAUACUUUGCCAGAAACUCAUG 908 15651 3UTR 2531 29.0% CAAAUACUUUGCCAGAAACUCAUGG 909 15652 3UTR 2537 78.0% CUUUGCCAGAAACUCAUGGAUGGCU 910 15653 3UTR 2538 52.4% UUUGCCAGAAACUCAUGGAUGGCUU 911 15654 3UTR 2540 59.7% UGCCAGAAACUCAUGGAUGGCUUAA 912 15655 3UTR 2541 45.1% GCCAGAAACUCAUGGAUGGCUUAAG 913 15656 3UTR 2542 42.1% CCAGAAACUCAUGGAUGGCUUAAGG 914 15657 3UTR 2543 76.9% CAGAAACUCAUGGAUGGCUUAAGGA 915 15658 3UTR 2544 29.0% AGAAACUCAUGGAUGGCUUAAGGAA 916 15659 3UTR 2547 45.2% AACUCAUGGAUGGCUUAAGGAACUU 917 15660 3UTR 2560 38.4% CUUAAGGAACUUGAACUCAAACGAG 918 15661 3UTR 2561 33.3% UUAAGGAACUUGAACUCAAACGAGC 919 15662 3UTR 2562 31.9% UAAGGAACUUGAACUCAAACGAGCC 920 15663 3UTR 2563 44.5% AAGGAACUUGAACUCAAACGAGCCA 921 15664 3UTR 2564 90.1% AGGAACUUGAACUCAAACGAGCCAG 922 15665 3UTR 2566 64.4% GAACUUGAACUCAAACGAGCCAGAA 923 15666 3UTR 2623 32.5% AAGUGAGUUAUUAUAUGACCGAGAA 924 15667 3UTR 2681 34.0% UGUUAUGUAUCAGCUGCCUAAGGAA 925 15668 3UTR 2791 59.0% UUUAAUUGUAAAUGGUUCUUUGUCA 926 15669 3UTR 2792 56.3% UUAAUUGUAAAUGGUUCUUUGUCAG 927 15670 3UTR 2793 46.8% UAAUUGUAAAUGGUUCUUUGUCAGU 928 15671 3UTR 2795 53.2% AUUGUAAAUGGUUCUUUGUCAGUUU 929 15672 3UTR 2798 33.1% GUAAAUGGUUCUUUGUCAGUUUAGU 930 15673 3UTR 2809 32.8% UUUGUCAGUUUAGUAAACCAGUGAA 931 15674 3UTR 2813 40.9% UCAGUUUAGUAAACCAGUGAAAUGU 932 15675 3UTR 2816 38.1% GUUUAGUAAACCAGUGAAAUGUUGA 933 15676 3UTR 2840 59.4% AAAUGUUUUGACAUGUACUGGUCAA 934 15677 3UTR 2957 77.9% UGGAUAUAGAAGCCAGCAUAAUUGA 935 15678 3UTR 2958 74.1% GGAUAUAGAAGCCAGCAUAAUUGAA 936 15679 3UTR 2959 52.4% GAUAUAGAAGCCAGCAUAAUUGAAA 937 15680 3UTR 2963 49.9% UAGAAGCCAGCAUAAUUGAAAACAC 938 15681 3UTR 2964 45.3% AGAAGCCAGCAUAAUUGAAAACACA 939 15682 3UTR 2966 45.5% AAGCCAGCAUAAUUGAAAACACAUC 940 15683 3UTR 3137 60.5% ACAAAUGUAUGUUUCUUUUAGCUGG 941 15684 3UTR 3138 63.6% CAAAUGUAUGUUUCUUUUAGCUGGC 942 15685 3UTR 3142 58.4% UGUAUGUUUCUUUUAGCUGGCCAGU 943 15686 3UTR 3144 56.3% UAUGUUUCUUUUAGCUGGCCAGUAC 944 15687 3UTR 3145 52.1% AUGUUUCUUUUAGCUGGCCAGUACU 945 15688 3UTR 3147 74.6% GUUUCUUUUAGCUGGCCAGUACUUU 946 15689 3UTR 3150 70.4% UCUUUUAGCUGGCCAGUACUUUUGA 947 15690 3UTR 3154 61.7% UUAGCUGGCCAGUACUUUUGAGUAA 948 15691 3UTR 3156 52.3% AGCUGGCCAGUACUUUUGAGUAAAG 949 15692 3UTR 3157 72.2% GCUGGCCAGUACUUUUGAGUAAAGC 950 15693 3UTR 3158 62.4% CUGGCCAGUACUUUUGAGUAAAGCC 951 15694 3UTR 3180 49.0% GCCCCUAUAGUUUGACUUGCACUAC 952 15695 3UTR 3182 43.9% CCCUAUAGUUUGACUUGCACUACAA 953 15696 3UTR 3183 35.2% CCUAUAGUUUGACUUGCACUACAAA 954 15697 3UTR 3184 38.1% CUAUAGUUUGACUUGCACUACAAAU 955 15698 3UTR 3185 73.3% UAUAGUUUGACUUGCACUACAAAUG 956 15699 3UTR 3256 86.3% UUCAUUAUUAUGACAUAAGCUACCU 957 15700 3UTR 3258 61.6% CAUUAUUAUGACAUAAGCUACCUGG 958 15701 3UTR 3342 66.0% UUCAUCUUCCAAGCAUCAUUACUAA 959 15702 3UTR 3346 67.3% UCUUCCAAGCAUCAUUACUAACCAA 960 15703 3UTR 3358 63.6% CAUUACUAACCAAGUCAGACGUUAA 961 15704 3UTR 3396 71.8% UAGGAAAAGGAGGAAUGUUAUAGAU 962 15705 3UTR 3550 69.1% UUGUUAUUACAAAGAGGACACUUCA 963 15706 3UTR 3657 72.3% GGGGAAAAAAGUCCAGGUCAGCAUA 964 15707 3UTR 3671 79.7% AGGUCAGCAUAAGUCAUUUUGUGUA 965 15708 3UTR 3779 57.5% UUUCUUUCCUCUGAGUGAGAGUUAU 966 15709 3UTR 3783 62.6% UUUCCUCUGAGUGAGAGUUAUCUAU 967 15710 3UTR 3932 61.3% UAAAAAUUAAUAGGCAAAGCAAUGG 968 15711 3UTR 3934 44.3% AAAAUUAAUAGGCAAAGCAAUGGAA 969 15712 3UTR 4034 68.7% UUUUUUGGAAUUUCCUGACCAUUAA 970 15713 3UTR 4058 50.6% AUUAAAGAAUUGGAUUUGCAAGUUU 971 15714 3UTR 4120 69.8% UAAACAGCCCUUGUGUUGGAUGUAA 972 15715 3UTR 4147 39.5% CAAUCCCAGAUUUGAGUGUGUGUUG 973 15716 3UTR 4148 62.2% AAUCCCAGAUUUGAGUGUGUGUUGA 974 15717 3UTR 4152 34.2% CCAGAUUUGAGUGUGUGUUGAUUAU 975 15718 3UTR 4273 38.0% GUCUUUCCUCAUGAAUGCACUGAUA 976 15719 3UTR 4460 48.5% UAUUUUUGUGUUAAUCAGCAGUACA 977 15720 3UTR 4482 37.1% ACAAUUUGAUCGUUGGCAUGGUUAA 978 15721 3UTR 4580 60.1% GUUUUUGUGGUGCUCUAGUGGUAAA 979 15722 3UTR 4583 50.6% UUUGUGGUGCUCUAGUGGUAAAUAA 980 15723 3UTR 4584 42.1% UUGUGGUGCUCUAGUGGUAAAUAAA 981 15724 3UTR 4642 91.3% UCAGUACCAUCAUCGAGUCUAGAAA 982 15725 3UTR 4737 90.4% UUCUCCCUUAAGGACAGUCACUUCA 983 15726 3UTR 4751 94.6% CAGUCACUUCAGAAGUCAUGCUUUA 984 15727 3UTR 4753 87.2% GUCACUUCAGAAGUCAUGCUUUAAA 985 15728 3UTR 4858 70.2% GUAAUUGUUUGAGAUUUAGUUUCCA 986 15729 3UTR 4963 81.2% CGCCAGGGCCAAAAGAACUGGUCUA 987 15730 3UTR 5177 81.4% CCAGACUCCUCAAACGAGUUGCCAA 988

TABLE 7 hTGFB2 sd-rxRNA Target Gene hTGFB2 SEQ ID Duplex ID Single Strand ID sd-rxRNA sequences NO 18570 17560 mU.A.mU.mU.mU.A.mU.mU.G.mU.G.mU.A.Chl 989 17562 P.mU.A.fC.A.fC.A.A.fU.A.A.A.fU.A*A*fC*fU*fC*A*C 990 18571 17561 mU.mU.A.mU.mU.mU.A.mU.mU.G.mU.G.mU.A.Chl 991 17562 P.mU.A.fC.A.fC.A.A.fU.A.A.A.fU.A*A*fC*fU*fC*A*C 992 18572 17563 A.mU.mC.A.G.mU.G.mU.mU.A.A.A.A.Chl 993 17565 P.mU.fU.fU.fU.A.A.fC.A.fC.fU.G.A.fU*G*A*A*fC*fC*A 994 18573 17564 mC.A.mU.mC.A.G.mU.G.mU.mU.A.A.A.A.Chl 995 17565 P.mU.fU.fU.fU.A.A.fC.A.fC.fU.G.A.fU*G*A*A*fC*fC*A 996 18574 17566 A.mU.G.G.mC.mU.mU.A.A.G.G.A.A.Chl 997 17568 P.mU.fU.fC.fC.fU.fU.A.A.G.fC.fC.A.fU*fC*fC*A*fU*G*A 998 18575 17567 G.A.mU.G.G.mC.mU.mU.A.A.G.G.A.A.Chl 999 17568 P.mU.fU.fC.fC.fU.fU.A.A.G.fC.fC.A.fU*fC*fC*A*fU*G*A 1000 18576 17569 mU.mU.G.mU.G.mU.mU.mC.mU.G.mU.mU.A.Chl 1001 17571 P.mU.A.A.fC.A.G.A.A.fC.A.fC.A.A*A*fC*fU*fU*fC*C 1002 18577 17570 mU.mU.mU.G.mU.G.mU.mU.mC.mU.G.mU.mU.A.Chl 1003 17571 P.mU.A.A.fC.A.G.A.A.fC.A.fC.A.A*A*fC*fU*fU*fC*C 1004 18578 17572 A.A.A.mU.A.mC.mU.mU.mU.G.mC.mC.A.Chl 1005 17574 P.mU.G.G.fC.A.A.A.G.fU.A.fU.fU.fU*G*G*fU*fC*fU*C 1006 18579 17573 mC.A.A.A.mU.A.mC.mU.mU.mU.G.mC.mC.A.Chl 1007 17574 P.mU.G.G.fC.A.A.A.G.fU.A.fU.fU.fU*G*G*fU*fC*fU*C 1008 18580 17575 mC.mU.mU.G.mC.A.mC.mU.A.mC.A.A.A.Chl 1009 17577 P.mU.fU.fU.G.fU.A.G.fU.G.fC.A.A.G*fU*fC*A*A*A*C 1010 18581 17576 A.mC.mU.mU.G.mC.A.mC.mU.A.mC.A.A.A.Chl 1011 17577 P.mU.fU.fU.G.fU.A.G.fU.G.fC.A.A.G*fU*fC*A*A*A*C 1012 18582 17578 G.A.A.mU.mU.mU.A.mU.mU.A.G.mU.A.Chl 1013 17580 P.mU.A.fC.fU.A.A.fU.A.A.A.fU.fU.fC*fU*fU*fC*fC*A*G 1014 18583 17579 A.G.A.A.mU.mU.mU.A.mU.mU.A.G.mU.A.Chl 1015 17580 P.mU.A.fC.fU.A.A.fU.A.A.A.fU.fU.fC*fU*fU*fC*fC*A*G 1016 18584 17581 mU.mU.G.mC.A.mC.mU.A.mC.A.A.A.A.Chl 1017 17583 P.mU.fU.fU.fU.G.fU.A.G.fU.G.fC.A.A*G*fU*fC*A*A*A 1018 18585 17582 mC.mU.mU.G.mC.A.mC.mU.A.mC.A.A.A.A.Chl 1019 17583 P.mU.fU.fU.fU.G.fU.A.G.fU.G.fC.A.A*G*fU*fC*A*A*A 1020 18586 17584 A.mU.A.A.A.A.mC.A.G.G.mU.G.A.Chl 1021 17586 P.mU.fC.A.fC.fC.fU.G.fU.fU.fU.fU.A.fU*fU*fU*fU*fC*fC*A 1022 18587 17585 A.A.mU.A.A.A.A.mC.A.G.G.mU.G.A.Chl 1023 17586 P.mU.fC.A.fC.fC.fU.G.fU.fU.fU.fU.A.fU*fU*fU*fU*fC*fC*A 1024 18588 17587 G.A.mC.A.A.mC.A.A.mC.A.A.mC.A.Chl 1025 17588 P.mU.G.fU.fU.G.fU.fU.G.fU.fU.G.fU.fC*G*fU*fU*G*fU*U 1026 18589 17589 A.mU.G.mC.mU.mU.G.mU.A.A.mC.A.A.Chl 1027 17590 P.mU.fU.G.fU.fU.A.fC.A.A.G.fC.A.fU*fC*A*fU*fC*G*U 1028 18590 17591 mC.A.G.A.A.A.mC.mU.mC.A.mU.G.A.Chl 1029 17592 P.mU.fC.A.fU.G.A.G.fU.fU.fU.fC.fU.G*G*fC*A*A*A*G 1030 18591 17593 G.mU.A.mU.mU.G.mC.mU.A.mU.G.mC.A.Chl 1031 17594 P.mU.G.fC.A.fU.A.G.fC.A.A.fU.A.fC*A*G*A*A*A*A 1032 18592 17595 mC.mC.A.G.A.A.A.mC.mU.mC.A.mU.A.Chl 1033 17596 P.mU.A.fU.G.A.G.fU.fU.fU.fC.fU.G.G*fC*A*A*A*G*U 1034 18593 17597 A.mC.mU.mC.A.A.A.mC.G.A.G.mC.A.Chl 1035 17598 P.mU.G.fC.fU.fC.G.fU.fU.fU.G.A.G.fU*fU*fC*A*A*G*U 1036 18594 17599 A.mU.A.mU.G.A.mC.mC.G.A.G.A.A.Chl 1037 17600 P.mU.fU.fC.fU.fC.G.G.fU.fC.A.fU.A.fU*A*A*fU*A*A*C 1038 18595 17601 mC.G.A.mC.G.A.mC.A.A.mC.G.A.A.Chl 1039 17602 P.mU.fU.fC.G.fU.fU.G.fU.fC.G.fU.fC.G*fU*fC*A*fU*fC*A 1040 18596 17603 G.mU.A.A.A.mC.mC.A.G.mU.G.A.A.Chl 1041 17604 P.mU.fU.fC.A.fC.fU.G.G.fU.fU.fU.A.fC*fU*A*A*A*fC*U 1042 18597 17605 mU.mU.G.mU.mC.A.G.mU.mU.mU.A.G.A.Chl 1043 17606 P.mU.fC.fU.A.A.A.fC.fU.G.A.fC.A.A*A*G*A*A*fC*C 1044 18598 17607 mU.mC.A.mU.mC.A.G.mU.G.mU.mU.A.A.Chl 1045 17608 P.mU.fU.A.A.fC.A.fC.fU.G.A.fU.G.A*A*fC*fC*A*A*G 1046 18599 17609 A.A.mC.mU.mC.A.A.A.mC.G.A.G.A.Chl 1047 17610 P.mU.fC.fU.fC.G.fU.fU.fU.G.A.G.fU.fU*fC*A*A*G*fU*U 1048 18600 17611 mC.G.A.mC.A.A.mC.A.A.mC.A.A.A.Chl 1049 17612 P.mU.fU.fU.G.fU.fU.G.fU.fU.G.fU.fC.G*fU*fU*G*fU*fU*C 1050 18601 17613 A.mC.G.A.mC.A.A.mC.G.A.mU.G.A.Chl 1051 17614 P.mU.fC.A.fU.fC.G.fU.fU.G.fU.fC.G.fU*fC*G*fU*fC*A*U 1052 18602 17615 G.mC.mU.G.mC.mC.mU.A.A.G.G.A.A.Chl 1053 17616 P.mU.fU.fC.fC.fU.fU.A.G.G.fC.A.G.fC*fU*G*A*fU*A*C 1054 18603 17617 A.mU.mU.mC.mU.A.mC.A.mU.mU.mU.mC.A.Chl 1055 17618 P.mU.G.A.A.A.fU.G.fU.A.G.A.A.fU*A*A*G*G*fC*C 1056 18604 17619 G.mU.G.mU.G.mU.mU.G.A.mU.mU.A.A.Chl 1057 17620 P.mU.fU.A.A.fU.fC.A.A.fC.A.fC.A.fC*A*fC*fU*fC*A*A 1058 Rat TGFB2 sd-rxRNA 18678 18604 mC.G.G.mU.G.A.mC.A.A.mU.G.A.A-chol 1061 18605 mU.fU.fC.A.fU.fU.G.fU.fC.A.fC.fC.G*fU*G*A*fU*fU*U 1062 18679 18606 mU.mU.G.mU.mC.mU.mC.G.G.mU.A.mU.A-chol 1063 18607 mU.A.fU.A.fC.fC.G.A.G.A.fC.A.A*A*G*G*G*A*A 1064 18680 18608 G.A.G.mU.mU.G.mU.A.mU.G.mU.A.A-chol 1065 18609 mU.fU.A.fC.A.fU.A.fC.A.A.fC.fU.fC*fC*A*fC*fU*G*A 1066 18681 18610 A.mU.mU.mU.G.mU.mU.A.G.mU.G.mU.A-chol 1067 18611 mU.A.fC.A.fC.fU.A.A.fC.A.A.A.fU*fU*fC*fU*fU*fC*C 1068 18682 18612 G.mC.A.A.G.mU.mC.mU.G.A.G.A.A-chol 1069 18613 mU.fU.fC.fU.fC.A.G.A.fC.fU.fU.G.fC*fU*fC*A*G*fU*U 1070 18683 18614 A.A.A.mU.mC.A.mC.G.G.mU.G.A.A-chol 1071 18615 mU.fU.fC.A.fC.fC.G.fU.G.A.fU.fU.fU*fU*fC*A*fU*fC*C 1072 18684 18616 A.A.A.mU.G.mC.A.G.mC.mU.A.A.A-chol 1073 18617 mU.fU.fU.A.G.fC.fU.G.fC.A.fU.fU.fU*A*fC*A*A*G*A 1074 18685 18618 mC.mU.mU.G.G.A.A.A.A.mU.A.A.A-chol 1075 18619 mU.fU.fU.A.fU.fU.fU.fU.fC.fC.A.A.G*G*G*fC*A*A*U 1076 18686 18620 mC.mC.mU.mU.mU.G.A.A.mU.A.A.A.A-chol 1077 18621 mU.fU.fU.fU.A.fU.fU.fC.A.A.A.G.G*fU*A*fC*fU*G*G 1078 18687 18622 A.A.mC.A.mC.A.mC.mU.G.mC.A.A.A-chol 1079 18623 mU.fU.fU.G.fC.A.G.fU.G.fU.G.fU.fU*fU*fU*fC*A*fU*C 1080 18688 18624 A.A.A.A.mC.A.mC.A.mC.mU.G.mC.A-chol 1081 18625 mU.G.fC.A.G.fU.G.fU.G.fU.fU.fU.fU*fC*A*fU*fC*A*U 1082 18689 18626 G.A.A.G.G.mC.mC.mU.G.mU.mU.A.A-chol 1083 18627 mU.fU.A.A.fC.A.G.G.fC.fC.fU.fU.fC*fU*G*G*A*fC*A 1084 18690 18628 mU.A.mU.mU.G.mC.mU.mC.mU.G.mC.A.A-chol 1085 18629 mU.fU.G.fC.A.G.A.G.fC.A.A.fU.A*fC*A*G*A*G*G 1086

TABLE 8 hSPP1 sd-rxRNA Target Gene hSPP1 SEQ ID Duplex ID Single Strand ID sd-rxRNA sequence NO 18538 17430 G.A.mU.G.A.A.mU.mC.mU.G.A.mU.A.Chl 1087 17432 P.mU.A.fU.fC.A.G.A.fU.fU.fC.A.fU.fC*A*G*A*A*fU*G 1088 18539 17431 mU.G.A.mU.G.A.A.mU.mC.mU.G.A.mU.A.Chl 1089 17432 P.mU.A.fU.fC.A.G.A.fU.fU.fC.A.fU.fC*A*G*A*A*fU*G 1090 18540 17433 A.mU.mU.mU.G.mC.mU.mU.mU.mU.G.mC.A.Chl 1091 17435 P.mU.G.fC.A.A.A.A.G.fC.A.A.A.fU*fC*A*fC*fU*G*fC 1092 18541 17434 G.A.mU.mU.mU.G.mC.mU.mU.mU.mU.G.mC.A.Chl 1093 17435 P.mU.G.fC.A.A.A.A.G.fC.A.A.A.fU*fC*A*fC*fU*G*fC 1094 18542 17436 G.mU.G.A.mU.mU.mU.G.mC.mU.mU.mU.AChl 1095 17438 P.mU.A.A.A.G.fC.A.A.A.fU.fC.A.fC*fU*G*fC*A*A*fU 1096 18543 17437 A.G.mU.G.A.mU.mU.mU.G.mC.mU.mU.mU.A.Chl 1097 17438 P.mU.A.A.A.G.fC.A.A.A.fU.fC.A.fC*fU*G*fC*A*A*fU 1098 18544 17439 A.A.mU.mU.mU.mC.G.mU.A.mU.mU.mU.A.Chl 1099 17441 P.mU.A.A.A.fU.A.fC.G.A.A.A.fU.fU*fU*fC*A*G*G*fU 1100 18545 17440 A.A.A.mU.mU.mU.mC.G.mU.A.mU.mU.mU.A.Chl 1101 17441 P.mU.A.A.A.fU.A.fC.G.A.A.A.fU.fU*fU*fC*A*G*G*fU 1102 18546 17442 mC.A.mC.A.G.mC.mC.A.mU.G.A.A.A.Chl 1103 17444 P.mU.fU.fU.C.A.fU.G.G.fC.fU.G.fU.G*A*A*A*fU*fU*fC 1104 18547 17443 mU.mC.A.mC.A.G.mC.mC.A.mU.G.A.A.A.Chl 1105 17444 P.mU.fU.fU.C.A.fU.G.G.fC.fU.G.fU.G*A*A*A*fU*fU*fC 1106 18548 17445 G.A.mU.mU.mU.G.mC.mU.mU.mU.mU.G.A.Chl 1107 17447 P.mU.fC.A.A.A.A.G.fC.A.A.A.fU.fC*A*fC*fU*G*fC*A 1108 18549 17446 mU.G.A.mU.mU.mU.G.mC.mU.mU.mU.mU.G.A.Chl 1109 17447 P.mU.fC.A.A.A.A.G.fC.A.A.A.fU.fC*A*fC*fU*G*fC*A 1110 18550 17448 mU.mU.G.mC.mU.mU.mU.mU.G.mC.mC.mU.A.Chl 1111 17450 P.mU.A.G.G.fC.A.A.A.A.G.fC.A.A*A*fU*fC*A*fC*U 1112 18551 17449 mU.mU.mU.G.mC.mU.mU.mU.mU.G.mC.mC.mU.A.Chl 1113 17450 P.mU.A.G.G.fC.A.A.A.A.G.fC.A.A*A*fU*fC*A*fC*U 1114 18552 17451 mU.mU.mU.mC.mU.mC.A.G.mU.mU.mU.A.A.Chl 1115 17452 P.mU.fU.A.A.A.fC.fU.G.A.G.A.A.A*G*A*A*G*fC*A 1116 18553 17453 mU.mU.G.mC.A.mU.mU.mU.A.G.mU.mC.A.Chl 1117 17454 P.mU.G.A.fC.fU.A.A.A.fU.G.fC.A.A*A*G*fU*G*A*G 1118 18554 17455 A.mC.mU.mU.mU.G.mC.A.mU.mU.mU.A.A.Chl 1119 17456 P.mU.fU.A.A.A.fU.G.fC.A.A.A.G.fU*G*A*G*A*A*A 1120 18555 17457 A.mU.mU.mU.A.G.mU.mC.A.A.A.A.A.Chl 1121 17458 P.mU.fU.fU.fU.fU.G.A.fC.fU.A.A.A.fU*G*fC*A*A*A*G 1122 18556 17459 mU.mU.mC.mU.mU.mU.mC.mU.mC.A.G.mU.A.Chl 1123 17460 P.mU.A.fC.fU.G.A.G.A.A.A.G.A.A*G*fC*A*fU*fU*fU 1124 18557 17461 mU.mC.mU.mU.mU.mC.mU.mC.A.G.mU.mU.A.Chl 1125 17462 P.mU.A.A.fC.fU.G.A.G.A.A.A.G.A*A*G*fC*A*fU*fU 1126 18558 17463 G.A.A.A.G.A.G.A.A.mC.A.mU.A.Chl 1127 17464 P.mU.A.fU.G.fU.fU.fC.fU.fC.fU.fU.fU.fC*A*fU*fU*fU*fU*G 1128 18559 17465 mC.mU.mU.mU.G.mC.A.mU.mU.mU.A.G.A.Chl 1129 17466 P.mU.fC.fU.A.A.A.fU.G.fC.A.A.A.G*fU*G*A*G*A*A 1130 18560 17467 mU.mU.mU.G.mC.A.mU.mU.mU.A.G.mU.A.Chl 1131 17468 P.mU.A.fC.fU.A.A.A.fU.G.fC.A.A.A*G*fU*G*A*G*A 1132 18561 17469 mC.mU.mC.A.mC.mU.mU.mU.G.mC.A.mU.A.Chl 1133 17470 P.mU.A.fU.G.fC.A.A.A.G.fU.G.A.G*A*A*A*fU*fU*G 1134 18562 17471 mU.mU.mC.mU.mC.A.mC.mU.mU.mU.G.mC.A.Chl 1135 17472 P.mU.G.fC.A.A.A.G.fU.G.A.G.A.A*A*fU*fU*G*fU*A 1136 18563 17473 mC.A.mC.mU.mC.mC.A.G.mU.mU.G.mU.A.Chl 1137 17474 P.mU.A.fC.A.A.fC.fU.G.G.A.G.fU.G*A*A*A*A*fC*U 1138 18564 17475 A.A.mU.G.A.A.A.G.A.G.A.A.A.Chl 1139 17476 P.mU.fU.fU.fC.fU.fC.fU.fU.fU.fC.A.fU.fU*fU*fU*G*fC*fU*A 1140 18565 17477 mU.G.mC.A.G.mU.G.A.mU.mU.mU.mG.A.Chl 1141 17478 P.mU.fC.A.A.A.fU.fC.A.fC.fU.G.fC.A*A*fU*fU*fC*fU*C 1142 18566 17479 mU.G.A.A.A.G.A.G.A.A.mC.A.A.Chl 1143 17480 P.mU.fU.G.fU.fU.fC.fU.fC.fU.fU.fU.fC.A*fU*fU*fU*fU*G*C 1144 18567 17481 A.mC.mC.mU.G.A.A.A.mU.mU.mU.mC.A.Chl 1145 17482 P.mU.G.A.A.A.fU.fU.fU.fC.A.G.G.fU*G*fU*fU*fU*A*U 1146 18568 17483 G.A.A.mU.mU.G.mC.A.G.mU.G.A.A.Chl 1147 17484 P.mU.fU.fC.A.fC.fU.G.fC.A.A.fU.fU.fC*fU*fC*A*fU*G*G 1148 18569 17485 G.G.mC.mU.G.A.mU.mU.mC.mU.G.G.A.Chl 1149 17486 P.mU.fC.fC.A.G.A.A.fU.fC.A.G.fC.fC*fU*G*fU*fU*fU*A 1150 Rat Targeting SPP1 18662 18630 G.mU.mU.mC.G.mU.mU.G.mU.mU.mU.mC.A-chol 1151 18631 P.mU.G.A.A.A.fC.A.A.fC.G.A.A.fC*fU*A*A*G*fC*U 1152 18663 18632 G.A.A.A.G.A.A.A.mU.A.G.A.A-chol 1153 18633 P.mU.fU.fC.fU.A.fU.fU.fU.fC.fU.fU.fU.fC*fU*fC*fC*A*fC*A 1154 18664 18634 G.mU.G.G.A.G.A.A.A.G.A.A.A-chol 1155 18635 P.mU.fU.fU.fC.fU.fU.fU.fC.fU.fC.fC.A.fC*A*fU*A*fC*A*U 1156 18665 18636 mC.mU.G.mU.G.mU.mC.A.mC.mU.A.mU.A-chol 1157 18637 P.mU.A.fU.A.G.fU.G.A.fC.A.fC.A.G*A*fC*fU*A*fU*U 1158 18666 18638 G.mU.mU.mU.mC.mU.mC.A.G.mU.mU.mC.A-chol 1159 18639 P.mU.G.A.A.fC.fU.G.A.G.A.A.A.fC*A*A*G*fC*A*G 1160 18667 18640 mU.A.mC.A.G.G.A.A.mC.A.G.mC.A-chol 1161 18641 P.mU.G.fC.fU.G.fU.fU.fC.fC.fU.G.fU.A*A*G*fU*fU*fU*G 1162 18668 18642 G.mC.A.G.G.mC.A.A.A.mC.mU.mU.A-chol 1163 18643 P.mU.A.A.G.fU.fU.fU.G.fC.fC.fU.G.fC*fC*fU*fC*fU*A*C 1164 18669 18644 A.A.mC.mU.mU.A.mC.A.G.G.A.A.A-chol 1165 18645 P.mU.fU.fU.fC.fC.fU.G.fU.A.A.G.fU.fU*fU*G*fC*fC*fU*G 1166 18670 18646 mC.A.mC.mU.G.mC.A.mU.mU.mU.mU.A.A-chol 1167 18647 P.mU.fU.A.A.A.A.fU.G.fC.A.G.fU.G*G*fC*fC*A*fU*U 1168 18671 18648 G.A.mC.A.mC.mC.A.mC.mU.G.mU.A.A-chol 1169 18649 P.mU.fU.A.fC.A.G.fU.G.G.fU.G.fU.fC*fU*G*fC*A*fU*G 1170 18672 18650 A.G.A.G.G.mC.A.G.G.mC.A.A.A-chol 1171 18651 P.mU.fU.fU.G.fC.fC.fU.G.fC.fC.fU.fC.fU*A*fC*A*fU*A*C 1172 18673 18652 mU.A.G.A.G.G.mC.A.G.G.mC.A.A-chol 1173 18653 P.mU.fU.G.fC.fC.fU.G.fC.fC.fU.fC.fU.A*fC*A*fU*A*fC*A 1174 18674 18654 G.A.G.A.G.mU.mU.mC.A.mU.mC.mU.A-chol 1175 18655 P.mU.A.G.A.fU.G.A.A.fC.fU.fC.fU.fC*fU*A*A*fU*fU*C 1176 18675 18656 mU.G.mU.G.A.A.mU.A.A.A.mU.mC.A-chol 1177 18657 P.mU.G.A.fU.fU.fU.A.fU.U.fC.A.fC.A*fC*fC*A*fC*A*A 1178 18676 18658 G.mU.G.A.A.mU.A.A.A.mU.mC.mU.A-chol 1179 18659 P.mU.A.G.A.fU.fU.fU.A.fU.fU.fC.A.fC*A*fC*fC*A*fC*A 1180 18677 18660 mU.G.A.AmU.A.A.A.mU.mC.mU.mU.A-chol 1181 18661 P.mU.A.A.G.A.fU.fU.fU.A.fU.U.fC.A*fC*A*fC*fC*A*C 1182

TABLE 9 Inhibition of gene expression with hSPP1 ori sequences Target Gene Gene SEQ ID hSPP1 A549 0.1 nM Duplex ID Region Ref Pos NO Sense Strand Sequence Activity 14840 5UTR/CDS 155 1183 AAGGAAAACUCACUACCAUGAGAAA  4.4% 14841 5UTR/CDS 161 1184 AACUCACUACCAUGAGAAUUGCAGA 2.46% 14842 5UTR/CDS 163 1185 CUCACUACCAUGAGAAUUGCAGUGA 20.54%  14843 5UTR/CDS 164 1186 UCACUACCAUGAGAAUUGCAGUGAA  2.8% 14844 CDS 168 1187 UACCAUGAGAAUUGCAGUGAUUUGA  3.6% 14845 CDS 169 1188 ACCAUGAGAAUUGCAGUGAUUUGCA  5.2% 14846 CDS 171 1189 CAUGAGAAUUGCAGUGAUUUGCUUA  0.8% 14847 CDS 172 1190 AUGAGAAUUGCAGUGAUUUGCUUUA 0.95% 14848 CDS 173 1191 UGAGAAUUGCAGUGAUUUGCUUUUA  3.2% 14849 CDS 174 1192 GAGAAUUGCAGUGAUUUGCUUUUGA 4.14% 14850 CDS 175 1193 AGAAUUGCAGUGAUUUGCUUUUGCA  2.9% 14851 CDS 176 1194 GAAUUGCAGUGAUUUGCUUUUGCCA 8.38% 14852 CDS 177 1195 AAUUGCAGUGAUUUGCUUUUGCCUA  4.6% 14853 CDS 180 1196 UGCAGUGAUUUGCUUUUGCCUCCUA 11.1% 14854 CDS 181 1197 GCAGUGAUUUGCUUUUGCCUCCUAA 10.87%  14855 CDS 182 1198 CAGUGAUUUGCUUUUGCCUCCUAGA  5.3% 14856 CDS 206 1199 GCAUCACCUGUGCCAUACCAGUUAA 15.29%  14857 CDS 208 1200 AUCACCUGUGCCAUACCAGUUAAAA 22.6% 14858 CDS 212 1201 CCUGUGCCAUACCAGUUAAACAGGA 13.3% 14859 CDS 215 1202 GUGCCAUACCAGUUAAACAGGCUGA 21.2% 14860 CDS 216 1203 UGCCAUACCAGUUAAACAGGCUGAA 20.24%  14861 CDS 220 1204 AUACCAGUUAAACAGGCUGAUUCUA 12.5% 14862 CDS 221 1205 UACCAGUUAAACAGGCUGAUUCUGA  9.9% 14863 CDS 222 1206 ACCAGUUAAACAGGCUGAUUCUGGA  3.9% 14864 CDS 225 1207 AGUUAAACAGGCUGAUUCUGGAAGA 20.48%  14865 CDS 226 1208 GUUAAACAGGCUGAUUCUGGAAGUA 10.7% 14866 CDS 227 1209 UUAAACAGGCUGAUUCUGGAAGUUA 22.75%  14867 CDS 228 1210 UAAACAGGCUGAUUCUGGAAGUUCA 0.26% 14868 CDS 234 1211 GGCUGAUUCUGGAAGUUCUGAGGAA 0.34% 14869 CDS 236 1212 CUGAUUCUGGAAGUUCUGAGGAAAA  4.4% 14870 CDS 238 1213 GAUUCUGGAAGUUCUGAGGAAAAGA  4.5% 14871 CDS 239 1214 AUUCUGGAAGUUCUGAGGAAAAGCA  7.5% 14872 CDS 240 1215 UUCUGGAAGUUCUGAGGAAAAGCAA 101.3%  14873 CDS 338 1216 CCCCACAGACCCUUCCAAGUAAGUA 48.3% 14874 CDS 340 1217 CCACAGACCCUUCCAAGUAAGUCCA 33.9% 14875 CDS 342 1218 ACAGACCCUUCCAAGUAAGUCCAAA 16.1% 14876 CDS 343 1219 CAGACCCUUCCAAGUAAGUCCAACA 38.7% 14877 CDS 345 1220 GACCCUUCCAAGUAAGUCCAACGAA 54.2% 14878 CDS 348 1221 CCUUCCAAGUAAGUCCAACGAAAGA 12.54%  14879 CDS 349 1222 CUUCCAAGUAAGUCCAACGAAAGCA 32.44%  14880 CDS 351 1223 UCCAAGUAAGUCCAACGAAAGCCAA 17.1% 14881 CDS 353 1224 CAAGUAAGUCCAACGAAAGCCAUGA 32.94%  14882 CDS 358 1225 AAGUCCAACGAAAGCCAUGACCACA 65.1% 14883 CDS 362 1226 CCAACGAAAGCCAUGACCACAUGGA 76.9% 14884 CDS 363 1227 CAACGAAAGCCAUGACCACAUGGAA 69.8% 14885 CDS 366 1228 CGAAAGCCAUGACCACAUGGAUGAA 78.02%  14886 CDS 372 1229 CCAUGACCACAUGGAUGAUAUGGAA 19.49%  14887 CDS 377 1230 ACCACAUGGAUGAUAUGGAUGAUGA 20.43%  14888 CDS 393 1231 GGAUGAUGAAGAUGAUGAUGACCAA 29.1% 14889 CDS 394 1232 GAUGAUGAAGAUGAUGAUGACCAUA 24.5% 14890 CDS 396 1233 UGAUGAAGAUGAUGAUGACCAUGUA 25.90%  14891 CDS 398 1234 AUGAAGAUGAUGAUGACCAUGUGGA 20.5% 14892 CDS 399 1235 UGAAGAUGAUGAUGACCAUGUGGAA  7.9% 14893 CDS 430 1236 GACUCCAUUGACUCGAACGACUCUA 21.6% 14894 CDS 431 1237 ACUCCAUUGACUCGAACGACUCUGA 13.5% 14895 CDS 432 1238 CUCCAUUGACUCGAACGACUCUGAA 12.33%  14896 CDS 435 1239 CAUUGACUCGAACGACUCUGAUGAA 42.5% 14897 CDS 440 1240 ACUCGAACGACUCUGAUGAUGUAGA 22.54%  14898 CDS 441 1241 CUCGAACGACUCUGAUGAUGUAGAA 17.4% 14899 CDS 442 1242 UCGAACGACUCUGAUGAUGUAGAUA 11.2% 14900 CDS 443 1243 CGAACGACUCUGAUGAUGUAGAUGA 20.7% 14901 CDS 445 1244 AACGACUCUGAUGAUGUAGAUGACA 27.1% 14902 CDS 449 1245 ACUCUGAUGAUGUAGAUGACACUGA 39.8% 14903 CDS 450 1246 CUCUGAUGAUGUAGAUGACACUGAA  9.6% 14904 CDS 451 1247 UCUGAUGAUGUAGAUGACACUGAUA 4.44% 14905 CDS 452 1248 CUGAUGAUGUAGAUGACACUGAUGA  8.7% 14906 CDS 453 1249 UGAUGAUGUAGAUGACACUGAUGAA 16.72%  14907 CDS 461 1250 UAGAUGACACUGAUGAUUCUCACCA 42.9% 14908 CDS 462 1251 AGAUGACACUGAUGAUUCUCACCAA 30.1% 14909 CDS 469 1252 ACUGAUGAUUCUCACCAGUCUGAUA  9.1% 14910 CDS 470 1253 CUGAUGAUUCUCACCAGUCUGAUGA 19.0% 14911 CDS 471 1254 UGAUGAUUCUCACCAGUCUGAUGAA 42.1% 14912 CDS 472 1255 GAUGAUUCUCACCAGUCUGAUGAGA 59.1% 14913 CDS 476 1256 AUUCUCACCAGUCUGAUGAGUCUCA 38.2% 14914 CDS 479 1257 CUCACCAGUCUGAUGAGUCUCACCA 34.1% 14915 CDS 480 1258 UCACCAGUCUGAUGAGUCUCACCAA 48.45%  14916 CDS 483 1259 CCAGUCUGAUGAGUCUCACCAUUCA  9.5% 14917 CDS 484 1260 CAGUCUGAUGAGUCUCACCAUUCUA 21.5% 14918 CDS 485 1261 AGUCUGAUGAGUCUCACCAUUCUGA 18.6% 14919 CDS 486 1262 GUCUGAUGAGUCUCACCAUUCUGAA 20.2% 14920 CDS 487 1263 UCUGAUGAGUCUCACCAUUCUGAUA 10.9% 14921 CDS 488 1264 CUGAUGAGUCUCACCAUUCUGAUGA 18.9% 14922 CDS 489 1265 UGAUGAGUCUCACCAUUCUGAUGAA 10.7% 14923 CDS 490 1266 GAUGAGUCUCACCAUUCUGAUGAAA 28.15%  14924 CDS 493 1267 GAGUCUCACCAUUCUGAUGAAUCUA 18.33%  14925 CDS 495 1268 GUCUCACCAUUCUGAUGAAUCUGAA 7.61% 14926 CDS 496 1269 UCUCACCAUUCUGAUGAAUCUGAUA 2.99% 14927 CDS 497 1270 CUCACCAUUCUGAUGAAUCUGAUGA 7.44% 14928 CDS 498 1271 UCACCAUUCUGAUGAAUCUGAUGAA  9.7% 14929 CDS 499 1272 CACCAUUCUGAUGAAUCUGAUGAAA 16.96%  14930 CDS 501 1273 CCAUUCUGAUGAAUCUGAUGAACUA 3.08% 14931 CDS 505 1274 UCUGAUGAAUCUGAUGAACUGGUCA 13.24%  14932 CDS 510 1275 UGAAUCUGAUGAACUGGUCACUGAA 3.16% 14933 CDS 550 1276 CCAGCAACCGAAGUUUUCACUCCAA 14.02%  14934 CDS 554 1277 CAACCGAAGUUUUCACUCCAGUUGA 3.10% 14935 CDS 555 1278 AACCGAAGUUUUCACUCCAGUUGUA 5.27% 14936 CDS 572 1279 CAGUUGUCCCCACAGUAGACACAUA 13.2% 14937 CDS 573 1280 AGUUGUCCCCACAGUAGACACAUAA 27.01%  14938 CDS 574 1281 GUUGUCCCCACAGUAGACACAUAUA 8.76% 14939 CDS 588 1282 AGACACAUAUGAUGGCCGAGGUGAA 14.04%  14940 CDS 589 1283 GACACAUAUGAUGGCCGAGGUGAUA 18.40%  14941 CDS 598 1284 GAUGGCCGAGGUGAUAGUGUGGUUA 12.50%  14942 CDS 601 1285 GGCCGAGGUGAUAGUGUGGUUUAUA 13.76%  14943 CDS 602 1286 GCCGAGGUGAUAGUGUGGUUUAUGA 5.34% 14944 CDS 603 1287 CCGAGGUGAUAGUGUGGUUUAUGGA 29.69%  14945 CDS 604 1288 CGAGGUGAUAGUGUGGUUUAUGGAA 33.34%  14946 CDS 606 1289 AGGUGAUAGUGUGGUUUAUGGACUA 17.50%  14947 CDS 608 1290 GUGAUAGUGUGGUUUAUGGACUGAA 45.90%  14948 CDS 609 1291 UGAUAGUGUGGUUUAUGGACUGAGA 22.0% 14949 CDS 610 1292 GAUAGUGUGGUUUAUGGACUGAGGA 19.93%  14950 CDS 611 1293 AUAGUGUGGUUUAUGGACUGAGGUA 17.34%  14951 CDS 615 1294 UGUGGUUUAUGGACUGAGGUCAAAA 5.60% 14952 CDS 617 1295 UGGUUUAUGGACUGAGGUCAAAAUA 25.74%  14953 CDS 618 1296 GGUUUAUGGACUGAGGUCAAAAUCA 17.63%  14954 CDS 619 1297 GUUUAUGGACUGAGGUCAAAAUCUA 3.45% 14955 CDS 621 1298 UUAUGGACUGAGGUCAAAAUCUAAA 18.03%  14956 CDS 622 1299 UAUGGACUGAGGUCAAAAUCUAAGA 20.98%  14957 CDS 623 1300 AUGGACUGAGGUCAAAAUCUAAGAA 20.60%  14958 CDS 624 1301 UGGACUGAGGUCAAAAUCUAAGAAA 26.73%  14959 CDS 625 1302 GGACUGAGGUCAAAAUCUAAGAAGA 7.45% 14960 CDS 626 1303 GACUGAGGUCAAAAUCUAAGAAGUA 14.1% 14961 CDS 629 1304 UGAGGUCAAAAUCUAAGAAGUUUCA 8.61% 14962 CDS 630 1305 GAGGUCAAAAUCUAAGAAGUUUCGA 19.07%  14963 CDS 631 1306 AGGUCAAAAUCUAAGAAGUUUCGCA 6.08% 14964 CDS 632 1307 GGUCAAAAUCUAAGAAGUUUCGCAA 19.82%  14965 CDS 636 1308 AAAAUCUAAGAAGUUUCGCAGACCA 21.55%  14966 CDS 637 1309 AAAUCUAAGAAGUUUCGCAGACCUA 30.20%  14967 CDS 638 1310 AAUCUAAGAAGUUUCGCAGACCUGA 18.23%  14968 CDS 686 1311 ACGAGGACAUCACCUCACACAUGGA 14.85%  14969 CDS 687 1312 CGAGGACAUCACCUCACACAUGGAA 28.04%  14970 CDS 689 1313 AGGACAUCACCUCACACAUGGAAAA 3.80% 14971 CDS 698 1314 CCUCACACAUGGAAAGCGAGGAGUA 7.67% 14972 CDS 703 1315 CACAUGGAAAGCGAGGAGUUGAAUA  5.8% 14973 CDS 704 1316 ACAUGGAAAGCGAGGAGUUGAAUGA  5.3% 14974 CDS 705 1317 CAUGGAAAGCGAGGAGUUGAAUGGA 24.47%  14975 CDS 718 1318 GAGUUGAAUGGUGCAUACAAGGCCA 26.39%  14976 CDS 785 1319 GCCGUGGGAAGGACAGUUAUGAAAA 7.60% 14977 CDS 786 1320 CCGUGGGAAGGACAGUUAUGAAACA 8.75% 14978 CDS 788 1321 GUGGGAAGGACAGUUAUGAAACGAA 8.34% 14979 CDS 790 1322 GGGAAGGACAGUUAUGAAACGAGUA 5.38% 14980 CDS 792 1323 GAAGGACAGUUAUGAAACGAGUCAA 11.45%  14981 CDS 794 1324 AGGACAGUUAUGAAACGAGUCAGCA 11.78%  14982 CDS 795 1325 GGACAGUUAUGAAACGAGUCAGCUA 10.69%  14983 CDS 797 1326 ACAGUUAUGAAACGAGUCAGCUGGA 54.58%  14984 CDS 798 1327 CAGUUAUGAAACGAGUCAGCUGGAA 33.9% 14985 CDS 846 1328 CCACAAGCAGUCCAGAUUAUAUAAA 24.1% 14986 CDS 850 1329 AAGCAGUCCAGAUUAUAUAAGCGGA 27.86%  14987 CDS 854 1330 AGUCCAGAUUAUAUAAGCGGAAAGA 24.29%  14988 CDS 855 1331 GUCCAGAUUAUAUAAGCGGAAAGCA 54.43%  14989 CDS 859 1332 AGAUUAUAUAAGCGGAAAGCCAAUA 71.49%  14990 CDS 860 1333 GAUUAUAUAAGCGGAAAGCCAAUGA 69.64%  14991 CDS 861 1334 AUUAUAUAAGCGGAAAGCCAAUGAA 38.82%  14992 CDS 862 1335 UUAUAUAAGCGGAAAGCCAAUGAUA 20.77%  14993 CDS 865 1336 UAUAAGCGGAAAGCCAAUGAUGAGA 21.79%  14994 CDS 866 1337 AUAAGCGGAAAGCCAAUGAUGAGAA 50.00%  14995 CDS 867 1338 UAAGCGGAAAGCCAAUGAUGAGAGA 11.67%  14996 CDS 870 1339 GCGGAAAGCCAAUGAUGAGAGCAAA 13.5% 14997 CDS 871 1340 CGGAAAGCCAAUGAUGAGAGCAAUA 15.49%  14998 CDS 872 1341 GGAAAGCCAAUGAUGAGAGCAAUGA 8.55% 14999 CDS 873 1342 GAAAGCCAAUGAUGAGAGCAAUGAA 12.12%  15000 CDS 875 1343 AAGCCAAUGAUGAGAGCAAUGAGCA 16.14%  15001 CDS 878 1344 CCAAUGAUGAGAGCAAUGAGCAUUA 31.71%  15002 CDS 879 1345 CAAUGAUGAGAGCAAUGAGCAUUCA 32.25%  15003 CDS 881 1346 AUGAUGAGAGCAAUGAGCAUUCCGA 6.97% 15004 CDS 883 1347 GAUGAGAGCAAUGAGCAUUCCGAUA 23.11%  15005 CDS 885 1348 UGAGAGCAAUGAGCAUUCCGAUGUA 5.53% 15006 CDS 890 1349 GCAAUGAGCAUUCCGAUGUGAUUGA 10.69%  15007 CDS 893 1350 AUGAGCAUUCCGAUGUGAUUGAUAA 4.12% 15008 CDS 894 1351 UGAGCAUUCCGAUGUGAUUGAUAGA 6.49% 15009 CDS 895 1352 GAGCAUUCCGAUGUGAUUGAUAGUA 29.12%  15010 CDS 897 1353 GCAUUCCGAUGUGAUUGAUAGUCAA 3.54% 15011 CDS 899 1354 AUUCCGAUGUGAUUGAUAGUCAGGA 6.05% 15012 CDS 901 1355 UCCGAUGUGAUUGAUAGUCAGGAAA 3.31% 15013 CDS 906 1356 UGUGAUUGAUAGUCAGGAACUUUCA 12.71%  15014 CDS 907 1357 GUGAUUGAUAGUCAGGAACUUUCCA 13.95%  15015 CDS 909 1358 GAUUGAUAGUCAGGAACUUUCCAAA 4.03% 15016 CDS 912 1359 UGAUAGUCAGGAACUUUCCAAAGUC 11.96%  15017 CDS 913 1360 GAUAGUCAGGAACUUUCCAAAGUCA 14.01%  15018 CDS 914 1361 AUAGUCAGGAACUUUCCAAAGUCAA 5.56% 15019 CDS 916 1362 AGUCAGGAACUUUCCAAAGUCAGCA 13.92%  15020 CDS 917 1363 GUCAGGAACUUUCCAAAGUCAGCCA 19.00%  15021 CDS 923 1364 AACUUUCCAAAGUCAGCCGUGAAUA 17.56%  15022 CDS 925 1365 CUUUCCAAAGUCAGCCGUGAAUUCA 19.58%  15023 CDS 926 1366 UUUCCAAAGUCAGCCGUGAAUUCCA 6.54% 15024 CDS 935 1367 UCAGCCGUGAAUUCCACAGCCAUGA 16.15%  15025 CDS 936 1368 CAGCCGUGAAUUCCACAGCCAUGAA 20.62%  15026 CDS 937 1369 AGCCGUGAAUUCCACAGCCAUGAAA 5.21% 15027 CDS 943 1370 GAAUUCCACAGCCAUGAAUUUCACA 31.14%  15028 CDS 944 1371 AAUUCCACAGCCAUGAAUUUCACAA 35.63%  15029 CDS 945 1372 AUUCCACAGCCAUGAAUUUCACAGA 23.96%  15030 CDS 946 1373 UUCCACAGCCAUGAAUUUCACAGCA 15.20%  15031 CDS 947 1374 UCCACAGCCAUGAAUUUCACAGCCA 19.45%  15032 CDS 950 1375 ACAGCCAUGAAUUUCACAGCCAUGA 25.74%  15033 CDS 952 1376 AGCCAUGAAUUUCACAGCCAUGAAA 2.59% 15034 CDS 953 1377 GCCAUGAAUUUCACAGCCAUGAAGA 6.00% 15035 CDS 954 1378 CCAUGAAUUUCACAGCCAUGAAGAA 4.60% 15036 CDS 956 1379 AUGAAUUUCACAGCCAUGAAGAUAA 9.20% 15037 CDS 957 1380 UGAAUUUCACAGCCAUGAAGAUAUA 10.84%  15038 CDS 958 1381 GAAUUUCACAGCCAUGAAGAUAUGA 40.20%  15039 CDS 959 1382 AAUUUCACAGCCAUGAAGAUAUGCA 37.25%  15040 CDS 960 1383 AUUUCACAGCCAUGAAGAUAUGCUA 8.21% 15041 CDS 961 1384 UUUCACAGCCAUGAAGAUAUGCUGA 12.01%  15042 CDS 964 1385 CACAGCCAUGAAGAUAUGCUGGUUA 12.25%  15043 CDS 983 1386 UGGUUGUAGACCCCAAAAGUAAGGA 19.65%  15044 CDS 984 1387 GGUUGUAGACCCCAAAAGUAAGGAA 28.19%  15045 CDS 985 1388 GUUGUAGACCCCAAAAGUAAGGAAA 17.92%  15046 CDS 986 1389 UUGUAGACCCCAAAAGUAAGGAAGA 7.94% 15047 CDS 987 1390 UGUAGACCCCAAAAGUAAGGAAGAA 15.09%  15048 CDS 988 1391 GUAGACCCCAAAAGUAAGGAAGAAA 20.01%  15049 CDS 989 1392 UAGACCCCAAAAGUAAGGAAGAAGA 7.25% 15050 CDS 990 1393 AGACCCCAAAAGUAAGGAAGAAGAA 12.42%  15051 CDS 995 1394 CCAAAAGUAAGGAAGAAGAUAAACA 8.96% 15052 CDS 996 1395 CAAAAGUAAGGAAGAAGAUAAACAA 6.85% 15053 CDS 997 1396 AAAAGUAAGGAAGAAGAUAAACACA 14.15%  15054 CDS 998 1397 AAAGUAAGGAAGAAGAUAAACACCA 12.32%  15055 CDS 999 1398 AAGUAAGGAAGAAGAUAAACACCUA 8.83% 15056 CDS 1001 1399 GUAAGGAAGAAGAUAAACACCUGAA 15.09%  15057 CDS 1002 1400 UAAGGAAGAAGAUAAACACCUGAAA 4.91% 15058 CDS 1007 1401 AAGAAGAUAAACACCUGAAAUUUCA 1.43% 15059 CDS 1008 1402 AGAAGAUAAACACCUGAAAUUUCGA 3.51% 15060 CDS 1009 1403 GAAGAUAAACACCUGAAAUUUCGUA 15.12%  15061 CDS 1010 1404 AAGAUAAACACCUGAAAUUUCGUAA 28.56%  15062 CDS 1013 1405 AUAAACACCUGAAAUUUCGUAUUUA 5.74% 15063 CDS 1015 1406 AAACACCUGAAAUUUCGUAUUUCUA 13.01%  15064 CDS 1024 1407 AAAUUUCGUAUUUCUCAUGAAUUAA 15.54%  15065 CDS 1030 1408 CGUAUUUCUCAUGAAUUAGAUAGUA 9.47% 15066 CDS 1031 1409 GUAUUUCUCAUGAAUUAGAUAGUGA 30.03%  15067 CDS 1032 1410 UAUUUCUCAUGAAUUAGAUAGUGCA 5.31% 15068 CDS 1036 1411 UCUCAUGAAUUAGAUAGUGCAUCUA 9.74% 15069 CDS 1037 1412 CUCAUGAAUUAGAUAGUGCAUCUUA 10.78%  15070 CDS 1038 1413 UCAUGAAUUAGAUAGUGCAUCUUCA 91.87%  15071 CDS 1039 1414 CAUGAAUUAGAUAGUGCAUCUUCUA 93.82%  15072 CDS 1040 1415 AUGAAUUAGAUAGUGCAUCUUCUGA 96.06%  15073 CDS 1041 1416 UGAAUUAGAUAGUGCAUCUUCUGAA 94.91%  15074 CDS 1042 1417 GAAUUAGAUAGUGCAUCUUCUGAGA 97.91%  15075 CDS 1043 1418 AAUUAGAUAGUGCAUCUUCUGAGGA 93.76%  15076 CDS 1044 1419 AUUAGAUAGUGCAUCUUCUGAGGUA 103.92%   15077 CDS 1045 1420 UUAGAUAGUGCAUCUUCUGAGGUCA 95.85%  15078 CDS/3UTR 1052 1421 GUGCAUCUUCUGAGGUCAAUUAAAA 93.83%  15079 CDS/3UTR 1053 1422 UGCAUCUUCUGAGGUCAAUUAAAAA 90.69%  15080 CDS/3UTR 1054 1423 GCAUCUUCUGAGGUCAAUUAAAAGA 101.49%   15081 CDS/3UTR 1055 1424 CAUCUUCUGAGGUCAAUUAAAAGGA 110.27%   15082 CDS/3UTR 1056 1425 AUCUUCUGAGGUCAAUUAAAAGGAA 99.36%  15083 CDS/3UTR 1057 1426 UCUUCUGAGGUCAAUUAAAAGGAGA 95.31%  15084 CDS/3UTR 1058 1427 CUUCUGAGGUCAAUUAAAAGGAGAA 15.55%  15085 3UTR 1081 1428 AAAAAAUACAAUUUCUCACUUUGCA 3.59% 15086 3UTR 1083 1429 AAAAUACAAUUUCUCACUUUGCAUU 3.46% 15087 3UTR 1086 1430 AUACAAUUUCUCACUUUGCAUUUAG 2.37% 15088 3UTR 1087 1431 UACAAUUUCUCACUUUGCAUUUAGU 3.54% 15089 3UTR 1088 1432 ACAAUUUCUCACUUUGCAUUUAGUC 2.85% 15090 3UTR 1089 1433 CAAUUUCUCACUUUGCAUUUAGUCA 2.35% 15091 3UTR 1093 1434 UUCUCACUUUGCAUUUAGUCAAAAG 1.38% 15092 3UTR 1125 1435 GCUUUAUAGCAAAAUGAAAGAGAAC 4.11% 15093 3UTR 1127 1436 UUUAUAGCAAAAUGAAAGAGAACAU 3.91% 15094 3UTR 1128 1437 UUAUAGCAAAAUGAAAGAGAACAUG 3.59% 15095 3UTR 1147 1438 AACAUGAAAUGCUUCUUUCUCAGUU 1.80% 15096 3UTR 1148 1439 ACAUGAAAUGCUUCUUUCUCAGUUU 2.17% 15097 3UTR 1150 1440 AUGAAAUGCUUCUUUCUCAGUUUAU 2.93% 15098 3UTR 1153 1441 AAAUGCUUCUUUCUCAGUUUAUUGG 2.18% 15099 3UTR 1154 1442 AAUGCUUCUUUCUCAGUUUAUUGGU 3.92% 15100 3UTR 1156 1443 UGCUUCUUUCUCAGUUUAUUGGUUG 4.08% 15101 3UTR 1157 1444 GCUUCUUUCUCAGUUUAUUGGUUGA 1.74% 15102 3UTR 1158 1445 CUUCUUUCUCAGUUUAUUGGUUGAA 4.74% 15103 3UTR 1159 1446 UUCUUUCUCAGUUUAUUGGUUGAAU 2.65% 15104 3UTR 1168 1447 AGUUUAUUGGUUGAAUGUGUAUCUA 2.57% 15105 3UTR 1178 1448 UUGAAUGUGUAUCUAUUUGAGUCUG 3.76% 15106 3UTR 1179 1449 UGAAUGUGUAUCUAUUUGAGUCUGG 2.91% 15107 3UTR 1183 1450 UGUGUAUCUAUUUGAGUCUGGAAAU 0.62% 15108 3UTR 1184 1451 GUGUAUCUAUUUGAGUCUGGAAAUA 2.45% 15109 3UTR 1186 1452 GUAUCUAUUUGAGUCUGGAAAUAAC 2.18% 15110 3UTR 1191 1453 UAUUUGAGUCUGGAAAUAACUAAUG 2.44% 15111 3UTR 1218 1454 UUUGAUAAUUAGUUUAGUUUGUGGC 19.35%  15112 3UTR 1219 1455 UUGAUAAUUAGUUUAGUUUGUGGCU 6.19% 15113 3UTR 1222 1456 AUAAUUAGUUUAGUUUGUGGCUUCA 3.25% 15114 3UTR 1224 1457 AAUUAGUUUAGUUUGUGGCUUCAUG 2.47% 15115 3UTR 1225 1458 AUUAGUUUAGUUUGUGGCUUCAUGG 2.28% 15116 3UTR 1226 1459 UUAGUUUAGUUUGUGGCUUCAUGGA 3.40% 15117 3UTR 1227 1460 UAGUUUAGUUUGUGGCUUCAUGGAA 4.12% 15118 3UTR 1244 1461 UCAUGGAAACUCCCUGUAAACUAAA 2.63% 15119 3UTR 1245 1462 CAUGGAAACUCCCUGUAAACUAAAA 2.20% 15120 3UTR 1246 1463 AUGGAAACUCCCUGUAAACUAAAAG 3.56% 15121 3UTR 1247 1464 UGGAAACUCCCUGUAAACUAAAAGC 3.73% 15122 3UTR 1248 1465 GGAAACUCCCUGUAAACUAAAAGCU 2.43% 15123 3UTR 1249 1466 GAAACUCCCUGUAAACUAAAAGCUU 2.28% 15124 3UTR 1251 1467 AACUCCCUGUAAACUAAAAGCUUCA 5.40% 15125 3UTR 1253 1468 CUCCCUGUAAACUAAAAGCUUCAGG 8.21% 15126 3UTR 1286 1469 UAUGUUCAUUCUAUAGAAGAAAUGC 3.17% 15127 3UTR 1294 1470 UUCUAUAGAAGAAAUGCAAACUAUC 2.45% 15128 3UTR 1295 1471 UCUAUAGAAGAAAUGCAAACUAUCA 3.97% 15129 3UTR 1296 1472 CUAUAGAAGAAAUGCAAACUAUCAC 3.86% 15130 3UTR 1297 1473 UAUAGAAGAAAUGCAAACUAUCACU 1.84% 15131 3UTR 1299 1474 UAGAAGAAAUGCAAACUAUCACUGU 2.53% 15132 3UTR 1302 1475 AAGAAAUGCAAACUAUCACUGUAUU 2.25% 15133 3UTR 1303 1476 AGAAAUGCAAACUAUCACUGUAUUU 3.32% 15134 3UTR 1357 1477 AUUUAUGUAGAAGCAAACAAAAUAC 1.86% 15135 3UTR 1465 1478 UAUCUUUUUGUGGUGUGAAUAAAUC 3.40% 15136 3UTR 1466 1479 AUCUUUUUGUGGUGUGAAUAAAUCU 3.49% 15137 3UTR 1467 1480 UCUUUUUGUGGUGUGAAUAAAUCUU 3.03% 15138 3UTR 1468 1481 CUUUUUGUGGUGUGAAUAAAUCUUU 3.62% 15139 3UTR 1496 1482 CUUGAAUGUAAUAAGAAUUUGGUGG 61.48%  15140 3UTR 1497 1483 UUGAAUGUAAUAAGAAUUUGGUGGU 71.54%  15141 3UTR 1504 1484 UAAUAAGAAUUUGGUGGUGUCAAUU 58.54%  15142 3UTR 1511 1485 AAUUUGGUGGUGUCAAUUGCUUAUU 56.93%  15143 3UTR 1512 1486 AUUUGGUGGUGUCAAUUGCUUAUUU 81.22%  15144 3UTR 1513 1487 UUUGGUGGUGUCAAUUGCUUAUUUG 59.16%  15145 3UTR 1514 1488 UUGGUGGUGUCAAUUGCUUAUUUGU 59.46%  15146 3UTR 1540 1489 UUCCCACGGUUGUCCAGCAAUUAAU 67.40% 

TABLE 10 hCTGF sd-rxRNA CTGF Target Gene Single Strand SEQ ID Duplex ID ID sd-rxRNA sequence NO 17356 17007 A.mC.A.mU.mU.A.A.mC.mU.mC.A.mU.A.Chl 1490 17009 P.mU.A.fU.G.A.G.fU.fU.A.A.fU.G.fU*fC*fU*fC*fU*fC*A 1491 17357 17008 G.A.mC.A.mU.mU.A.A.mC.mU.mC.A.mU.A.Chl 1492 17009 P.mU.A.fU.G.A.G.fU.fU.A.A.fU.G.fU*fC*fU*fC*fU*fC*A 1493 17358 17010 mU.G.A.A.G.A.A.mU.G.mU.mU.A.A.Chl 1494 17012 P.mU.fU.A.A.fC.A.fU.fU.fC.fU.fU.fC.A*A*A*fC*fC*A*G 1495 17359 17011 mU.mU.G.A.A.G.A.A.mU.G.mU.mU.A.A.Chl 1496 17012 P.mU.fU.A.A.fC.A.fU.fU.fC.fU.fU.fC.A*A*A*fC*fC*A*G 1497 17360 17013 G.A.mU.A.G.mC.A.mU.mC.mU.mU.A.A.Chl 1498 17015 P.mU.fU.A.A.G.A.fU.G.fC.fU.A.fU.fC*fU*G*A*fU*G*A 1499 17361 17014 A.G.A.mU.A.G.mC.A.mU.mC.mU.mU.A.A.Chl 1500 17015 P.mU.fU.A.A.G.A.fU.G.fC.fU.A.fU.fC*fU*G*A*fU*G*A 1501 17362 17016 mU.G.A.A.G.mU.G.mU.A.A.mU.mU.A.Chl 1502 17017 P.mU.A.A.fU.fU.A.fC.A.fC.fU.fU.fC.A*A*A*fU*A*G*C 1503 17363 17018 A.A.mU.mU.G.A.G.A.A.G.G.A.A.Chl 1504 17019 P.mU.fU.fC.fC.fU.fU.fC.fU.fC.A.A.fU.fU*A*fC*A*fC*fU*U 1505 17364 17020 mU.mU.G.A.G.A.A.G.G.A.A.A.A.Chl 1506 17021 P.mU.fU.fU.fU.fC.fC.fU.fU.fC.fU.fC.A.A*fU*fU*A*fC*A*C 1507 17365 17022 mC.A.mU.mU.mC.mU.G.A.mU.mU.mC.G.A.Chl 1508 17023 P.mU.fC.G.A.A.fU.fC.A.G.A.A.fU.G*fU*fC*A*G*A*G 1509 17366 17024 mU.mU.mC.mU.G.A.mU.mU.mC.G.A.A.A.Chl 1510 17025 P.mU.fU.fU.fC.G.A.A.fU.fC.A.G.A.A*fU*G*fU*fC*A*G 1511 17367 17026 mC.mU.G.mU.mC.G.A.mU.mU.A.G.A.A.Chl 1512 17027 P.mU.fU.fC.fU.A.A.fU.fC.G.A.fC.A.G*G*A*fU*fU*fC*C 1513 17368 17028 mU.mU.mU.G.mC.mC.mU.G.mU.A.A.mC.A.Chl 1514 17030 P.mU.G.fU.fU.A.fC.A.G.G.fC.A.A.A*fU*fU*fC*A*fC*U 1515 17369 17029 A.mU.mU.mU.G.mC.mC.mU.G.mU.A.A.mC.A.Chl 1516 17030 P.mU.G.fU.fU.A.fC.A.G.G.fC.A.A.A*fU*fU*fC*A*fC*U 1517 17370 17031 A.mC.A.A.G.mC.mC.A.G.A.mU.mU.A.Chl 1518 17033 P.mU.A.A.fU.fC.fU.G.G.fC.fU.fU.G.fU*fU*A*fC*A*G*G 1519 17371 17032 “A.A.mC.A.A.G.mC.mC.A.G.A.mU.mU.A.Chl 1520 17033 P.mU.A.A.fU.fC.fU.G.G.fC.fU.fU.G.fU*fU*A*fC*A*G*G 1521 17372 17034 mC.A.G.mU.mU.mU.A.mU.mU.mU.G.mU.A.Chl 1522 17035 P.mU.A.fC.A.A.A.fU.A.A.A.fC.fU.G*fU*fC*fC*G*A*A 1523 17373 17036 mU.G.mU.mU.G.A.G.A.G.mU.G.mU.A.Chl 1524 17038 P.mU.A.fC.A.fC.fU.fC.fU.fC.A.A.fC.A*A*A*fU*A*A*A 1525 17374 17037 mU.mU.G.mU.mU.G.A.G.A.G.mU.G.mU.A.Chl 1526 17038 P.mU.A.fC.A.fC.fU.fC.fU.fC.A.A.fC.A*A*A*fU*A*A*A 1527 17375 17039 mU.G.mC.A.mC.mC.mU.mU.mU.mC.mU.A.A.Chl 1528 17041 P.mU.fU.A.G.A.A.A.G.G.fU.G.fC.A*A*A*fC*A*fU*G 1529 17376 17040 mU.mU.G.mC.A.mC.mC.mU.mU.mU.mC.mU.A.A.Chl 1530 17041 P.mU.fU.A.G.A.A.A.G.G.fU.G.fC.A*A*A*fC*A*fU*G 1531 17377 17042 mU.mU.G.A.G.mC.mU.mU.mU.mC.mU.G.A.Chl 1532 17043 P.mU.fC.A.G.A.A.A.G.fC.fU.fC.A.A*A*fC*fU*fU*G*A 1533 17378 17044 mU.G.A.G.A.G.mU.G.mU.G.A.mC.A.Chl 1534 17045 P.mU.G.fU.fC.A.fC.A.fC.fU.fC.fU.fC.A*A*fC*A*A*A*U 1535 17379 17046 A.G.mU.G.mU.G.A.mC.mC.A.A.A.A.Chl 1536 17048 P.mU.fU.fU.fU.G.G.fU.fC.A.fC.A.fC.fU*fC*fU*fC*A*A*C 1537 17380 17047 G.A.G.mU.G.mU.G.A.mC.mC.A.A.A.A.Chl 1538 17048 P.mU.fU.fU.fU.G.G.fU.fC.A.fC.A.fC.fU*fC*fU*fC*A*A*C 1539 17381 17049 G.mU.G.mU.G.A.mC.mC.A.A.A.A.A.Chl 1540 17050 P.mU.fU.fU.fU.fU.G.G.fU.fC.A.fC.A.fC*fU*fC*fU*fC*A*A 1541 17382 17051 mU.G.mU.G.A.mC.mC.A.A.A.A.G.A.Chl 1542 17053 P.mU.fC.fU.fU.fU.fU.G.G.fU.fC.A.fC.A*fC*fU*fC*fU*fC*A 1543 17383 17052 G.mU.G.mU.G.A.mC.mC.A.A.A.A.G.A.Chl 1544 17053 P.mU.fC.fU.fU.fU.fU.G.G.fU.fC.A.fC.A*fC*fU*fC*fU*fC*A 1545 17384 17054 G.mU.G.A.mC.mC.A.A.A.A.G.mU.A.Chl 1546 17055 P.mU.A.fC.fU.fU.fU.fU.G.G.fU.fC.A.fC*A*fC*fU*fC*fU*C 1547 17385 17056 G.A.mC.mC.A.A.A.A.G.mU.mU.A.A.Chl 1548 17057 P.mU.fU.A.A.fC.fU.fU.fU.fU.G.G.fU.fC*A*fC*A*fC*fU*C 1549 17386 17058 G.mC.A.mC.mC.mU.mU.mU.mC.mU.A.G.A.Chl 1550 17059 P.mU.fC.fU.A.G.A.A.A.G.G.fU.G.fC*A*A*A*fC*A*U 1551 17387 17060 mC.mC.mU.mU.mU.mC.mU.A.G.mU.mU.G.A.Chl 1552 17061 P.mU.fC.A.A.fC.fU.A.G.A.A.A.G.G*fU*G*fC*A*A*A 1553

TABLE 11 Inhibition of gene expression with hCTGF ori sequences Target Gene Gene Ref SEQ ID CTGF % Expression Duplex Name Region Pos NO Sense sequence (0.1 nM) 14542 CDS 774 1554 UUUGGCCCAGACCCAACUAUGAUUA 96% 14543 CDS 776 1555 UGGCCCAGACCCAACUAUGAUUAGA 94% 14544 CDS 785 1556 CCCAACUAUGAUUAGAGCCAACUGA 55% 14545 CDS 786 1557 CCAACUAUGAUUAGAGCCAACUGCA 89% 14546 CDS 934 1558 CUUGCGAAGCUGACCUGGAAGAGAA 63% 14547 CDS 938 1559 CGAAGCUGACCUGGAAGAGAACAUA 70% 14548 CDS 940 1560 AAGCUGACCUGGAAGAGAACAUUAA 65% 14549 CDS 941 1561 AGCUGACCUGGAAGAGAACAUUAAA 81% 14550 CDS 943 1562 CUGACCUGGAAGAGAACAUUAAGAA 85% 14551 CDS 944 1563 UGACCUGGAAGAGAACAUUAAGAAA 61% 14552 CDS 945 1564 GACCUGGAAGAGAACAUUAAGAAGA 73% 14553 CDS 983 1565 CCGUACUCCCAAAAUCUCCAAGCCA 86% 14554 CDS 984 1566 CGUACUCCCAAAAUCUCCAAGCCUA 64% 14555 CDS 985 1567 GUACUCCCAAAAUCUCCAAGCCUAA 71% 14556 CDS 986 1568 UACUCCCAAAAUCUCCAAGCCUAUA 71% 14557 CDS 987 1569 ACUCCCAAAAUCUCCAAGCCUAUCA 84% 14558 CDS 988 1570 CUCCCAAAAUCUCCAAGCCUAUCAA 64% 14559 CDS 989 1571 UCCCAAAAUCUCCAAGCCUAUCAAA 64% 14560 CDS 990 1572 CCCAAAAUCUCCAAGCCUAUCAAGA 87% 14561 CDS 1002 1573 AAGCCUAUCAAGUUUGAGCUUUCUA 46% 14562 CDS 1003 1574 AGCCUAUCAAGUUUGAGCUUUCUGA 30% 14563 CDS 1004 1575 GCCUAUCAAGUUUGAGCUUUCUGGA 63% 14564 CDS 1008 1576 AUCAAGUUUGAGCUUUCUGGCUGCA 77% 14565 CDS 1025 1577 UGGCUGCACCAGCAUGAAGACAUAA 96% 14566 CDS 1028 1578 CUGCACCAGCAUGAAGACAUACCGA 79% 14567 CDS 1029 1579 UGCACCAGCAUGAAGACAUACCGAA 58% 14568 CDS 1033 1580 CCAGCAUGAAGACAUACCGAGCUAA 59% 14569 CDS 1035 1581 AGCAUGAAGACAUACCGAGCUAAAA 76% 14570 CDS 1036 1582 GCAUGAAGACAUACCGAGCUAAAUA 71% 14571 CDS 1050 1583 CGAGCUAAAUUCUGUGGAGUAUGUA 73% 14572 CDS 1051 1584 GAGCUAAAUUCUGUGGAGUAUGUAA 72% 14573 CDS 1053 1585 GCUAAAUUCUGUGGAGUAUGUACCA 87% 14574 CDS 1054 1586 CUAAAUUCUGUGGAGUAUGUACCGA 83% 14575 CDS 1135 1587 CUGACGGCGAGGUCAUGAAGAAGAA 77% 14576 CDS 1138 1588 ACGGCGAGGUCAUGAAGAAGAACAA 72% 14577 CDS 1139 1589 CGGCGAGGUCAUGAAGAAGAACAUA 85% 14578 CDS 1143 1590 GAGGUCAUGAAGAAGAACAUGAUGA 83% 14579 CDS 1145 1591 GGUCAUGAAGAAGAACAUGAUGUUA 91% 14580 CDS 1148 1592 CAUGAAGAAGAACAUGAUGUUCAUA 92% 14581 CDS 1157 1593 GAACAUGAUGUUCAUCAAGACCUGA 84% 14582 CDS 1161 1594 AUGAUGUUCAUCAAGACCUGUGCCA 92% 14583 CDS 1203 1595 GGAGACAAUGACAUCUUUGAAUCGA 62% 14584 CDS 1204 1596 GAGACAAUGACAUCUUUGAAUCGCA 56% 14585 CDS 1205 1597 AGACAAUGACAUCUUUGAAUCGCUA 30% 14586 CDS 1206 1598 GACAAUGACAUCUUUGAAUCGCUGA 47% 14587 CDS 1207 1599 ACAAUGACAUCUUUGAAUCGCUGUA 29% 14588 CDS 1208 1600 CAAUGACAUCUUUGAAUCGCUGUAA 50% 14589 CDS 1209 1601 AAUGACAUCUUUGAAUCGCUGUACA 39% 14590 CDS 1210 1602 AUGACAUCUUUGAAUCGCUGUACUA 44% 14591 CDS 1211 1603 UGACAUCUUUGAAUCGCUGUACUAA 39% 14592 CDS 1212 1604 GACAUCUUUGAAUCGCUGUACUACA 55% 14593 CDS 1213 1605 ACAUCUUUGAAUCGCUGUACUACAA 59% 14594 CDS 1216 1606 UCUUUGAAUCGCUGUACUACAGGAA 80% 14595 CDS 1217 1607 CUUUGAAUCGCUGUACUACAGGAAA 80% 14596 CDS 1223 1608 AUCGCUGUACUACAGGAAGAUGUAA 59% 14597 CDS 1224 1609 UCGCUGUACUACAGGAAGAUGUACA 62% 14598 CDS 1239 1610 AAGAUGUACGGAGACAUGGCAUGAA 59% 14599 CDS 1253 1611 CAUGGCAUGAAGCCAGAGAGUGAGA 65% 14600 3UTR 1266 1612 CAGAGAGUGAGAGACAUUAACUCAA 43% 14601 3UTR 1267 1613 AGAGAGUGAGAGACAUUAACUCAUA 25% 14602 3UTR 1268 1614 GAGAGUGAGAGACAUUAACUCAUUA 33% 14603 3UTR 1269 1615 AGAGUGAGAGACAUUAACUCAUUAA 42% 14604 3UTR 1270 1616 GAGUGAGAGACAUUAACUCAUUAGA 28% 14605 3UTR 1271 1617 AGUGAGAGACAUUAACUCAUUAGAA 34% 14606 3UTR 1272 1618 GUGAGAGACAUUAACUCAUUAGACA 30% 14607 3UTR 1273 1619 UGAGAGACAUUAACUCAUUAGACUA 33% 14608 3UTR 1275 1620 AGAGACAUUAACUCAUUAGACUGGA 42% 14609 3UTR 1277 1621 AGACAUUAACUCAUUAGACUGGAAA 25% 14610 3UTR 1278 1622 GACAUUAACUCAUUAGACUGGAACA 31% 14611 3UTR 1279 1623 ACAUUAACUCAUUAGACUGGAACUA 32% 14612 3UTR 1281 1624 AUUAACUCAUUAGACUGGAACUUGA 23% 14613 3UTR 1284 1625 AACUCAUUAGACUGGAACUUGAACA 39% 14614 3UTR 1285 1626 ACUCAUUAGACUGGAACUUGAACUA 30% 14615 3UTR 1286 1627 CUCAUUAGACUGGAACUUGAACUGA 43% 14616 3UTR 1287 1628 UCAUUAGACUGGAACUUGAACUGAA 26% 14617 3UTR 1291 1629 UAGACUGGAACUUGAACUGAUUCAA 33% 14618 3UTR 1293 1630 GACUGGAACUUGAACUGAUUCACAA 43% 14619 3UTR 1294 1631 ACUGGAACUUGAACUGAUUCACAUA 28% 14620 3UTR 1295 1632 CUGGAACUUGAACUGAUUCACAUCA 41% 14621 3UTR 1296 1633 UGGAACUUGAACUGAUUCACAUCUA 34% 14622 3UTR 1298 1634 GAACUUGAACUGAUUCACAUCUCAA 31% 14623 3UTR 1299 1635 AACUUGAACUGAUUCACAUCUCAUA 31% 14624 3UTR 1300 1636 ACUUGAACUGAUUCACAUCUCAUUA 33% 14625 3UTR 1301 1637 CUUGAACUGAUUCACAUCUCAUUUA 28% 14626 3UTR 1326 1638 UCCGUAAAAAUGAUUUCAGUAGCAA 30% 14627 3UTR 1332 1639 AAAAUGAUUUCAGUAGCACAAGUUA 28% 14628 3UTR 1395 1640 CCCAAUUCAAAACAUUGUGCCAUGA 63% 14629 3UTR 1397 1641 CAAUUCAAAACAUUGUGCCAUGUCA 39% 14630 3UTR 1402 1642 CAAAACAUUGUGCCAUGUCAAACAA 34% 14631 3UTR 1408 1643 AUUGUGCCAUGUCAAACAAAUAGUA 33% 14632 3UTR 1409 1644 UUGUGCCAUGUCAAACAAAUAGUCA 33% 14633 3UTR 1412 1645 UGCCAUGUCAAACAAAUAGUCUAUA 36% 14634 3UTR 1416 1646 AUGUCAAACAAAUAGUCUAUCAACA 30% 14635 3UTR 1435 1647 UCAACCCCAGACACUGGUUUGAAGA 39% 14636 3UTR 1436 1648 CAACCCCAGACACUGGUUUGAAGAA 47% 14637 3UTR 1438 1649 ACCCCAGACACUGGUUUGAAGAAUA 45% 14638 3UTR 1439 1650 CCCCAGACACUGGUUUGAAGAAUGA 40% 14639 3UTR 1442 1651 CAGACACUGGUUUGAAGAAUGUUAA 21% 14640 3UTR 1449 1652 UGGUUUGAAGAAUGUUAAGACUUGA 22% 14641 3UTR 1453 1653 UUGAAGAAUGUUAAGACUUGACAGA 24% 14642 3UTR 1454 1654 UGAAGAAUGUUAAGACUUGACAGUA 37% 14643 3UTR 1462 1655 GUUAAGACUUGACAGUGGAACUACA 20% 14644 3UTR 1470 1656 UUGACAGUGGAACUACAUUAGUACA 30% 14645 3UTR 1471 1657 UGACAGUGGAACUACAUUAGUACAA 43% 14646 3UTR 1474 1658 CAGUGGAACUACAUUAGUACACAGA 36% 14647 3UTR 1475 1659 AGUGGAACUACAUUAGUACACAGCA 38% 14648 3UTR 1476 1660 GUGGAACUACAUUAGUACACAGCAA 35% 14649 3UTR 1477 1661 UGGAACUACAUUAGUACACAGCACA 34% 14650 3UTR 1478 1662 GGAACUACAUUAGUACACAGCACCA 33% 14651 3UTR 1479 1663 GAACUACAUUAGUACACAGCACCAA 39% 14652 3UTR 1480 1664 AACUACAUUAGUACACAGCACCAGA 27% 14653 3UTR 1481 1665 ACUACAUUAGUACACAGCACCAGAA 29% 14654 3UTR 1482 1666 CUACAUUAGUACACAGCACCAGAAA 38% 14655 3UTR 1483 1667 UACAUUAGUACACAGCACCAGAAUA 28% 14656 3UTR 1484 1668 ACAUUAGUACACAGCACCAGAAUGA 31% 14657 3UTR 1486 1669 AUUAGUACACAGCACCAGAAUGUAA 26% 14658 3UTR 1487 1670 UUAGUACACAGCACCAGAAUGUAUA 31% 14659 3UTR 1489 1671 AGUACACAGCACCAGAAUGUAUAUA 35% 14660 3UTR 1490 1672 GUACACAGCACCAGAAUGUAUAUUA 34% 14661 3UTR 1497 1673 GCACCAGAAUGUAUAUUAAGGUGUA 32% 14662 3UTR 1503 1674 GAAUGUAUAUUAAGGUGUGGCUUUA 42% 14663 3UTR 1539 1675 AGGGUACCAGCAGAAAGGUUAGUAA 28% 14664 3UTR 1543 1676 UACCAGCAGAAAGGUUAGUAUCAUA 29% 14665 3UTR 1544 1677 ACCAGCAGAAAGGUUAGUAUCAUCA 33% 14666 3UTR 1548 1678 GCAGAAAGGUUAGUAUCAUCAGAUA 34% 14667 3UTR 1557 1679 UUAGUAUCAUCAGAUAGCAUCUUAA 22% 14668 3UTR 1576 1680 UCUUAUACGAGUAAUAUGCCUGCUA 48% 14669 3UTR 1577 1681 CUUAUACGAGUAAUAUGCCUGCUAA 31% 14670 3UTR 1579 1682 UAUACGAGUAAUAUGCCUGCUAUUA 43% 14671 3UTR 1580 1683 AUACGAGUAAUAUGCCUGCUAUUUA 39% 14672 3UTR 1581 1684 UACGAGUAAUAUGCCUGCUAUUUGA 33% 14673 3UTR 1582 1685 ACGAGUAAUAUGCCUGCUAUUUGAA 40% 14674 3UTR 1584 1686 GAGUAAUAUGCCUGCUAUUUGAAGA 38% 14675 3UTR 1585 1687 AGUAAUAUGCCUGCUAUUUGAAGUA 24% 14676 3UTR 1586 1688 GUAAUAUGCCUGCUAUUUGAAGUGA 34% 14677 3UTR 1587 1689 UAAUAUGCCUGCUAUUUGAAGUGUA 26% 14678 3UTR 1589 1690 AUAUGCCUGCUAUUUGAAGUGUAAA 26% 14679 3UTR 1591 1691 AUGCCUGCUAUUUGAAGUGUAAUUA 25% 14680 3UTR 1596 1692 UGCUAUUUGAAGUGUAAUUGAGAAA 36% 14681 3UTR 1599 1693 UAUUUGAAGUGUAAUUGAGAAGGAA 22% 14682 3UTR 1600 1694 AUUUGAAGUGUAAUUGAGAAGGAAA 22% 14683 3UTR 1601 1695 UUUGAAGUGUAAUUGAGAAGGAAAA 19% 14684 3UTR 1609 1696 GUAAUUGAGAAGGAAAAUUUUAGCA 53% 14685 3UTR 1610 1697 UAAUUGAGAAGGAAAAUUUUAGCGA 55% 14686 3UTR 1611 1698 AAUUGAGAAGGAAAAUUUUAGCGUA 20% 14687 3UTR 1612 1699 AUUGAGAAGGAAAAUUUUAGCGUGA 23% 14688 3UTR 1613 1700 UUGAGAAGGAAAAUUUUAGCGUGCA 37% 14689 3UTR 1614 1701 UGAGAAGGAAAAUUUUAGCGUGCUA 31% 14690 3UTR 1619 1702 AGGAAAAUUUUAGCGUGCUCACUGA 46% 14691 3UTR 1657 1703 CCAGUGACAGCUAGGAUGUGCAUUA 42% 14692 3UTR 1661 1704 UGACAGCUAGGAUGUGCAUUCUCCA 39% 14693 3UTR 1682 1705 UCCAGCCAUCAAGAGACUGAGUCAA 53% 14694 3UTR 1685 1706 AGCCAUCAAGAGACUGAGUCAAGUA 71% 14695 3UTR 1686 1707 GCCAUCAAGAGACUGAGUCAAGUUA 54% 14696 3UTR 1687 1708 CCAUCAAGAGACUGAGUCAAGUUGA 71% 14697 3UTR 1688 1709 CAUCAAGAGACUGAGUCAAGUUGUA 74% 14698 3UTR 1689 1710 AUCAAGAGACUGAGUCAAGUUGUUA 61% 14699 3UTR 1690 1711 UCAAGAGACUGAGUCAAGUUGUUCA 59% 14700 3UTR 1691 1712 CAAGAGACUGAGUCAAGUUGUUCCA 73% 14701 3UTR 1692 1713 AAGAGACUGAGUCAAGUUGUUCCUA 78% 14702 3UTR 1693 1714 AGAGACUGAGUCAAGUUGUUCCUUA 60% 14703 3UTR 1695 1715 AGACUGAGUCAAGUUGUUCCUUAAA 63% 14704 3UTR 1696 1716 GACUGAGUCAAGUUGUUCCUUAAGA 92% 14705 3UTR 1697 1717 ACUGAGUCAAGUUGUUCCUUAAGUA 74% 14706 3UTR 1707 1718 GUUGUUCCUUAAGUCAGAACAGCAA 70% 14707 3UTR 1724 1719 AACAGCAGACUCAGCUCUGACAUUA 69% 14708 3UTR 1725 1720 ACAGCAGACUCAGCUCUGACAUUCA 67% 14709 3UTR 1726 1721 CAGCAGACUCAGCUCUGACAUUCUA 71% 14710 3UTR 1727 1722 AGCAGACUCAGCUCUGACAUUCUGA 73% 14711 3UTR 1728 1723 GCAGACUCAGCUCUGACAUUCUGAA 60% 14712 3UTR 1729 1724 CAGACUCAGCUCUGACAUUCUGAUA 72% 14713 3UTR 1732 1725 ACUCAGCUCUGACAUUCUGAUUCGA 24% 14714 3UTR 1733 1726 CUCAGCUCUGACAUUCUGAUUCGAA 32% 14715 3UTR 1734 1727 UCAGCUCUGACAUUCUGAUUCGAAA 23% 14716 3UTR 1735 1728 CAGCUCUGACAUUCUGAUUCGAAUA 27% 14717 3UTR 1736 1729 AGCUCUGACAUUCUGAUUCGAAUGA 38% 14718 3UTR 1739 1730 UCUGACAUUCUGAUUCGAAUGACAA 28% 14719 3UTR 1741 1731 UGACAUUCUGAUUCGAAUGACACUA 29% 14720 3UTR 1742 1732 GACAUUCUGAUUCGAAUGACACUGA 33% 14721 3UTR 1743 1733 ACAUUCUGAUUCGAAUGACACUGUA 28% 14722 3UTR 1747 1734 UCUGAUUCGAAUGACACUGUUCAGA 39% 14723 3UTR 1748 1735 CUGAUUCGAAUGACACUGUUCAGGA 36% 14724 3UTR 1750 1736 GAUUCGAAUGACACUGUUCAGGAAA 33% 14725 3UTR 1751 1737 AUUCGAAUGACACUGUUCAGGAAUA 30% 14726 3UTR 1759 1738 GACACUGUUCAGGAAUCGGAAUCCA 34% 14727 3UTR 1760 1739 ACACUGUUCAGGAAUCGGAAUCCUA 35% 14728 3UTR 1761 1740 CACUGUUCAGGAAUCGGAAUCCUGA 40% 14729 3UTR 1768 1741 CAGGAAUCGGAAUCCUGUCGAUUAA 34% 14730 3UTR 1769 1742 AGGAAUCGGAAUCCUGUCGAUUAGA 31% 14731 3UTR 1770 1743 GGAAUCGGAAUCCUGUCGAUUAGAA 24% 14732 3UTR 1771 1744 GAAUCGGAAUCCUGUCGAUUAGACA 32% 14733 3UTR 1772 1745 AAUCGGAAUCCUGUCGAUUAGACUA 29% 14734 3UTR 1774 1746 UCGGAAUCCUGUCGAUUAGACUGGA 34% 14735 3UTR 1777 1747 GAAUCCUGUCGAUUAGACUGGACAA 51% 14736 3UTR 1782 1748 CUGUCGAUUAGACUGGACAGCUUGA 88% 14737 3UTR 1783 1749 UGUCGAUUAGACUGGACAGCUUGUA 38% 14738 3UTR 1797 1750 GACAGCUUGUGGCAAGUGAAUUUGA 46% 14739 3UTR 1798 1751 ACAGCUUGUGGCAAGUGAAUUUGCA 52% 14740 3UTR 1800 1752 AGCUUGUGGCAAGUGAAUUUGCCUA 43% 14741 3UTR 1801 1753 GCUUGUGGCAAGUGAAUUUGCCUGA 51% 14742 3UTR 1802 1754 CUUGUGGCAAGUGAAUUUGCCUGUA 32% 14743 3UTR 1803 1755 UUGUGGCAAGUGAAUUUGCCUGUAA 31% 14744 3UTR 1804 1756 UGUGGCAAGUGAAUUUGCCUGUAAA 29% 14745 3UTR 1805 1757 GUGGCAAGUGAAUUUGCCUGUAACA 20% 14746 3UTR 1806 1758 UGGCAAGUGAAUUUGCCUGUAACAA 34% 14747 3UTR 1807 1759 GGCAAGUGAAUUUGCCUGUAACAAA 31% 14748 3UTR 1808 1760 GCAAGUGAAUUUGCCUGUAACAAGA 27% 14749 3UTR 1809 1761 CAAGUGAAUUUGCCUGUAACAAGCA 34% 14750 3UTR 1810 1762 AAGUGAAUUUGCCUGUAACAAGCCA 36% 14751 3UTR 1811 1763 AGUGAAUUUGCCUGUAACAAGCCAA 31% 14752 3UTR 1814 1764 GAAUUUGCCUGUAACAAGCCAGAUA 24% 14753 3UTR 1815 1765 AAUUUGCCUGUAACAAGCCAGAUUA 21% 14754 3UTR 1816 1766 AUUUGCCUGUAACAAGCCAGAUUUA 22% 14755 3UTR 1910 1767 AAGUUAAUUUAAAGUUGUUUGUGCA 58% 14756 3UTR 1911 1768 AGUUAAUUUAAAGUUGUUUGUGCCA 73% 14757 3UTR 1912 1769 GUUAAUUUAAAGUUGUUUGUGCCUA 64% 14758 3UTR 1957 1770 UUUGAUAUUUCAAUGUUAGCCUCAA 42% 14759 3UTR 1961 1771 AUAUUUCAAUGUUAGCCUCAAUUUA 30% 14760 3UTR 1971 1772 GUUAGCCUCAAUUUCUGAACACCAA 34% 14761 3UTR 1974 1773 AGCCUCAAUUUCUGAACACCAUAGA 35% 14762 3UTR 1975 1774 GCCUCAAUUUCUGAACACCAUAGGA 33% 14763 3UTR 1976 1775 CCUCAAUUUCUGAACACCAUAGGUA 39% 14764 3UTR 1977 1776 CUCAAUUUCUGAACACCAUAGGUAA 27% 14765 3UTR 1978 1777 UCAAUUUCUGAACACCAUAGGUAGA 31% 14766 3UTR 1979 1778 CAAUUUCUGAACACCAUAGGUAGAA 49% 14767 3UTR 1980 1779 AAUUUCUGAACACCAUAGGUAGAAA 46% 14768 3UTR 1981 1780 AUUUCUGAACACCAUAGGUAGAAUA 40% 14769 3UTR 1982 1781 UUUCUGAACACCAUAGGUAGAAUGA 47% 14770 3UTR 1985 1782 CUGAACACCAUAGGUAGAAUGUAAA 33% 14771 3UTR 1986 1783 UGAACACCAUAGGUAGAAUGUAAAA 35% 14772 3UTR 1987 1784 GAACACCAUAGGUAGAAUGUAAAGA 31% 14773 3UTR 1988 1785 AACACCAUAGGUAGAAUGUAAAGCA 30% 14774 3UTR 1989 1786 ACACCAUAGGUAGAAUGUAAAGCUA 32% 14775 3UTR 1991 1787 ACCAUAGGUAGAAUGUAAAGCUUGA 31% 14776 3UTR 1992 1788 CCAUAGGUAGAAUGUAAAGCUUGUA 34% 14777 3UTR 1993 1789 CAUAGGUAGAAUGUAAAGCUUGUCA 31% 14778 3UTR 1994 1790 AUAGGUAGAAUGUAAAGCUUGUCUA 28% 14779 3UTR 1996 1791 AGGUAGAAUGUAAAGCUUGUCUGAA 32% 14780 3UTR 2002 1792 AAUGUAAAGCUUGUCUGAUCGUUCA 34% 14781 3UTR 2017 1793 UGAUCGUUCAAAGCAUGAAAUGGAA 31% 14782 3UTR 2021 1794 CGUUCAAAGCAUGAAAUGGAUACUA 39% 14783 3UTR 2022 1795 GUUCAAAGCAUGAAAUGGAUACUUA 25% 14784 3UTR 2023 1796 UUCAAAGCAUGAAAUGGAUACUUAA 22% 14785 3UTR 2047 1797 UAUGGAAAUUCUGCUCAGAUAGAAA 39% 14786 3UTR 2048 1798 AUGGAAAUUCUGCUCAGAUAGAAUA 35% 14787 3UTR 2059 1799 GCUCAGAUAGAAUGACAGUCCGUCA 44% 14788 3UTR 2060 1800 CUCAGAUAGAAUGACAGUCCGUCAA 41% 14789 3UTR 2062 1801 CAGAUAGAAUGACAGUCCGUCAAAA 46% 14790 3UTR 2063 1802 AGAUAGAAUGACAGUCCGUCAAAAA 45% 14791 3UTR 2065 1803 AUAGAAUGACAGUCCGUCAAAACAA 41% 14792 3UTR 2067 1804 AGAAUGACAGUCCGUCAAAACAGAA 36% 14793 3UTR 2068 1805 GAAUGACAGUCCGUCAAAACAGAUA 40% 14794 3UTR 2113 1806 AGUGUCCUUGGCAGGCUGAUUUCUA 42% 14795 3UTR 2114 1807 GUGUCCUUGGCAGGCUGAUUUCUAA 42% 14796 3UTR 2118 1808 CCUUGGCAGGCUGAUUUCUAGGUAA 111% 14797 3UTR 2127 1809 GCUGAUUUCUAGGUAGGAAAUGUGA 44% 14798 3UTR 2128 1810 CUGAUUUCUAGGUAGGAAAUGUGGA 44% 14799 3UTR 2130 1811 GAUUUCUAGGUAGGAAAUGUGGUAA 46% 14800 3UTR 2131 1812 AUUUCUAGGUAGGAAAUGUGGUAGA 45% 14801 3UTR 2142 1813 GGAAAUGUGGUAGCCUCACUUUUAA 37% 14802 3UTR 2146 1814 AUGUGGUAGCCUCACUUUUAAUGAA 39% 14803 3UTR 2149 1815 UGGUAGCCUCACUUUUAAUGAACAA 40% 14804 3UTR 2154 1816 GCCUCACUUUUAAUGAACAAAUGGA 35% 14805 3UTR 2155 1817 CCUCACUUUUAAUGAACAAAUGGCA 41% 14806 3UTR 2181 1818 UUAUUAAAAACUGAGUGACUCUAUA 26% 14807 3UTR 2182 1819 UAUUAAAAACUGAGUGACUCUAUAA 29% 14808 3UTR 2183 1820 AUUAAAAACUGAGUGACUCUAUAUA 28% 14809 3UTR 2186 1821 AAAAACUGAGUGACUCUAUAUAGCA 31% 14810 3UTR 2187 1822 AAAACUGAGUGACUCUAUAUAGCUA 28% 14811 3UTR 2188 1823 AAACUGAGUGACUCUAUAUAGCUGA 38% 14812 3UTR 2189 1824 AACUGAGUGACUCUAUAUAGCUGAA 44% 14813 3UTR 2190 1825 ACUGAGUGACUCUAUAUAGCUGAUA 38% 14814 3UTR 2255 1826 ACUGUUUUUCGGACAGUUUAUUUGA 29% 14815 3UTR 2256 1827 CUGUUUUUCGGACAGUUUAUUUGUA 25% 14816 3UTR 2263 1828 UCGGACAGUUUAUUUGUUGAGAGUA 29% 14817 3UTR 2265 1829 GGACAGUUUAUUUGUUGAGAGUGUA 24% 14818 3UTR 2268 1830 CAGUUUAUUUGUUGAGAGUGUGACA 26% 14819 3UTR 2269 1831 AGUUUAUUUGUUGAGAGUGUGACCA 37% 14820 3UTR 2272 1832 UUAUUUGUUGAGAGUGUGACCAAAA 27% 14821 3UTR 2273 1833 UAUUUGUUGAGAGUGUGACCAAAAA 30% 14822 3UTR 2274 1834 AUUUGUUGAGAGUGUGACCAAAAGA 26% 14823 3UTR 2275 1835 UUUGUUGAGAGUGUGACCAAAAGUA 27% 14824 3UTR 2276 1836 UUGUUGAGAGUGUGACCAAAAGUUA 30% 14825 3UTR 2277 1837 UGUUGAGAGUGUGACCAAAAGUUAA 29% 14826 3UTR 2278 1838 GUUGAGAGUGUGACCAAAAGUUACA 33% 14827 3UTR 2279 1839 UUGAGAGUGUGACCAAAAGUUACAA 35% 14828 3UTR 2281 1840 GAGAGUGUGACCAAAAGUUACAUGA 36% 14829 3UTR 2282 1841 AGAGUGUGACCAAAAGUUACAUGUA 36% 14830 3UTR 2283 1842 GAGUGUGACCAAAAGUUACAUGUUA 33% 14831 3UTR 2284 1843 AGUGUGACCAAAAGUUACAUGUUUA 31% 14832 3UTR 2285 1844 GUGUGACCAAAAGUUACAUGUUUGA 22% 14833 3UTR 2286 1845 UGUGACCAAAAGUUACAUGUUUGCA 40% 14834 3UTR 2293 1846 AAAAGUUACAUGUUUGCACCUUUCA 24% 14835 3UTR 2295 1847 AAGUUACAUGUUUGCACCUUUCUAA 23% 14836 3UTR 2296 1848 AGUUACAUGUUUGCACCUUUCUAGA 29% 14837 3UTR 2299 1849 UACAUGUUUGCACCUUUCUAGUUGA 27% 14838 3UTR 2300 1850 ACAUGUUUGCACCUUUCUAGUUGAA 29% 14839 3UTR 2301 1851 CAUGUUUGCACCUUUCUAGUUGAAA 35% Key: PS * = Phosphothioate Backbone Linkage RNA G = Guanine RNA U = Uracil RNA C = Cytosine RNA A = Adenine m 2′ Omethyl f 2′-Fluoro Phosphate P 5′ Phosphate

TABLE 12 Inhibition of gene expression with CTGF ori sequences (Accession Number: NM_001901.2) 25-mer Sense Strand (position 25 of SS, replaced with A) Oligo Gene Ref SEQ ID 25-mer Sense Strand (position 25 of SS, A549 0.1 nM ID Region Pos NO original base, not replaced by A) Activity 13843 CDS 1047 1852 UACCGAGCUAAAUUCUGUGGAGUAU 113% 13844 3UTR 2164 1853 UAAUGAACAAAUGGCCUUUAUUAAA 61% 13845 3UTR 1795 1854 UGGACAGCUUGUGGCAAGUGAAUUU 99% 13846 CDS 1228 1855 UGUACUACAGGAAGAUGUACGGAGA 87% 13847 CDS 1146 1856 GUCAUGAAGAAGAACAUGAUGUUCA 98% 13848 CDS 1150 1857 UGAAGAAGAACAUGAUGUUCAUCAA 105% 13849 CDS 1218 1858 UUUGAAUCGCUGUACUACAGGAAGA 91% 13850 3UTR 2262 1859 UUCGGACAGUUUAUUUGUUGAGAGU 50% 13851 CDS 1147 1860 UCAUGAAGAAGAACAUGAUGUUCAU 104% 13852 3UTR 2163 1861 UUAAUGAACAAAUGGCCUUUAUUAA 54% 13853 3UTR 1414 1862 CCAUGUCAAACAAAUAGUCUAUCAA 35% 13854 CDS 1195 1863 ACUGUCCCGGAGACAAUGACAUCUU 103% 13855 3UTR 1788 1864 AUUAGACUGGACAGCUUGUGGCAAG 103% 13856 3UTR 1793 1865 ACUGGACAGCUUGUGGCAAGUGAAU 81% 13857 3UTR 1891 1866 UAUAUAUGUACAGUUAUCUAAGUUA 73% 13858 3UTR 2270 1867 GUUUAUUUGUUGAGAGUGUGACCAA 76% 13859 482 1868 CAAGAUCGGCGUGUGCACCGCCAAA 95% 13860 CDS 942 1869 GCUGACCUGGAAGAGAACAUUAAGA 93% 13861 CDS 1199 1870 UCCCGGAGACAAUGACAUCUUUGAA 83% 13862 3UTR 2258 1871 GUUUUUCGGACAGUUUAUUUGUUGA 40% 13863 CDS 1201 1872 CCGGAGACAAUGACAUCUUUGAAUC 123% 13864 CDS 543 1873 CGCAGCGGAGAGUCCUUCCAGAGCA 124% 13865 3UTR 1496 1874 AGCACCAGAAUGUAUAUUAAGGUGU 109% 13866 CDS 793 1875 UGAUUAGAGCCAACUGCCUGGUCCA 125% 13867 CDS 1198 1876 GUCCCGGAGACAAUGACAUCUUUGA 64% 13868 3UTR 2160 1877 CUUUUAAUGAACAAAUGGCCUUUAU 68% 13869 CDS 1149 1878 AUGAAGAAGAACAUGAUGUUCAUCA 107% 13870 CDS 1244 1879 GUACGGAGACAUGGCAUGAAGCCAG 107% 13871 3UTR 1495 1880 CAGCACCAGAAUGUAUAUUAAGGUG 77% 13872 475 1881 CCAACCGCAAGAUCGGCGUGUGCAC 113% 13873 CDS 806 1882 CUGCCUGGUCCAGACCACAGAGUGG 113% 13874 CDS 819 1883 ACCACAGAGUGGAGCGCCUGUUCCA 99% 13875 CDS 1221 1884 GAAUCGCUGUACUACAGGAAGAUGU 97% 13876 CDS 1152 1885 AAGAAGAACAUGAUGUUCAUCAAGA 121% 13877 CDS 1163 1886 GAUGUUCAUCAAGACCUGUGCCUGC 125% 13878 3UTR 1494 1887 ACAGCACCAGAAUGUAUAUUAAGGU 94% 13879 3UTR 1890 1888 AUAUAUAUGUACAGUUAUCUAAGUU 94% 13880 473 1889 GGCCAACCGCAAGAUCGGCGUGUGC 122% 13881 544 1890 GCAGCGGAGAGUCCUUCCAGAGCAG 111% 13882 CDS 883 1891 ACAACGCCUCCUGCAGGCUAGAGAA 105% 13883 CDS 1240 1892 AGAUGUACGGAGACAUGGCAUGAAG 99% 13884 CDS 1243 1893 UGUACGGAGACAUGGCAUGAAGCCA 116% 13885 3UTR 2266 1894 GACAGUUUAUUUGUUGAGAGUGUGA 53% 13886 CDS 1011 1895 AAGUUUGAGCUUUCUGGCUGCACCA 118% 13887 CDS 1020 1896 CUUUCUGGCUGCACCAGCAUGAAGA 110% 13888 CDS 1168 1897 UCAUCAAGACCUGUGCCUGCCAUUA 119% 13889 1415 1898 CAUGUCAAACAAAUAGUCUAUCAAC 64% 13890 3UTR 1792 1899 GACUGGACAGCUUGUGGCAAGUGAA 53% 13891 3UTR 2156 1900 CUCACUUUUAAUGAACAAAUGGCCU 119% 13892 379 1901 GCUGCCGCGUCUGCGCCAAGCAGCU 112% 13893 CDS 1229 1902 GUACUACAGGAAGAUGUACGGAGAC 112% 13894 3UTR 1791 1903 AGACUGGACAGCUUGUGGCAAGUGA 65% 13895 3UTR 2158 1904 CACUUUUAAUGAACAAAUGGCCUUU 76% 13896 488 1905 CGGCGUGUGCACCGCCAAAGAUGGU 89% 13897 CDS 1151 1906 GAAGAAGAACAUGAUGUUCAUCAAG 119% 13898 CDS 1156 1907 AGAACAUGAUGUUCAUCAAGACCUG 125% 13899 CDS 1237 1908 GGAAGAUGUACGGAGACAUGGCAUG 114% 13900 CDS 1202 1909 CGGAGACAAUGACAUCUUUGAAUCG 130% 13901 CDS 1236 1910 AGGAAGAUGUACGGAGACAUGGCAU 135% 13902 3UTR 1786 1911 CGAUUAGACUGGACAGCUUGUGGCA 119% 13903 3UTR 1789 1912 UUAGACUGGACAGCUUGUGGCAAGU 108% 13904 3UTR 2290 1913 ACCAAAAGUUACAUGUUUGCACCUU 90% 13905 CDS 1017 1914 GAGCUUUCUGGCUGCACCAGCAUGA 121% 13906 CDS 1197 1915 UGUCCCGGAGACAAUGACAUCUUUG 125% 13907 CDS 1219 1916 UUGAAUCGCUGUACUACAGGAAGAU 98% 13908 3UTR 2159 1917 ACUUUUAAUGAACAAAUGGCCUUUA 52% 13909 486 1918 AUCGGCGUGUGCACCGCCAAAGAUG 119% 13910 CDS 826 1919 AGUGGAGCGCCUGUUCCAAGACCUG 139% 13911 CDS 1022 1920 UUCUGGCUGCACCAGCAUGAAGACA 144% 13912 3UTR 1492 1921 ACACAGCACCAGAAUGUAUAUUAAG 99% 13913 3UTR 1781 1922 CCUGUCGAUUAGACUGGACAGCUUG 89% 13914 485 1923 GAUCGGCGUGUGCACCGCCAAAGAU 131% 13915 CDS 1007 1924 UAUCAAGUUUGAGCUUUCUGGCUGC 92% 13916 CDS 1242 1925 AUGUACGGAGACAUGGCAUGAAGCC 106% 13917 3UTR 1787 1926 GAUUAGACUGGACAGCUUGUGGCAA 104% 13918 3UTR 1889 1927 UAUAUAUAUGUACAGUUAUCUAAGU 78% 13919 3UTR 2294 1928 AAAGUUACAUGUUUGCACCUUUCUA 28% 13920 CDS 821 1929 CACAGAGUGGAGCGCCUGUUCCAAG 108% 13921 CDS 884 1930 CAACGCCUCCUGCAGGCUAGAGAAG 125% 13922 3UTR 2260 1931 UUUUCGGACAGUUUAUUUGUUGAGA 43% 13923 CDS 889 1932 CCUCCUGCAGGCUAGAGAAGCAGAG 95% 13924 CDS 1226 1933 GCUGUACUACAGGAAGAUGUACGGA 122% 13925 3UTR 1493 1934 CACAGCACCAGAAUGUAUAUUAAGG 88% 13926 3UTR 1799 1935 CAGCUUGUGGCAAGUGAAUUUGCCU 89% 13927 CDS 807 1936 UGCCUGGUCCAGACCACAGAGUGGA 101% 13928 CDS 1107 1937 ACCACCCUGCCGGUGGAGUUCAAGU 113% 13929 CDS 1155 1938 AAGAACAUGAUGUUCAUCAAGACCU 109% 13930 CDS 1169 1939 CAUCAAGACCUGUGCCUGCCAUUAC 89% 13931 CDS 1241 1940 GAUGUACGGAGACAUGGCAUGAAGC 96% 13932 3UTR 1794 1941 CUGGACAGCUUGUGGCAAGUGAAUU 73% 13933 3UTR 1888 1942 AUAUAUAUAUGUACAGUUAUCUAAG 98% 13934 3UTR 2289 1943 GACCAAAAGUUACAUGUUUGCACCU 77% 13935 373 1944 GCGGCUGCUGCCGCGUCUGCGCCAA 85% 13936 CDS 799 1945 GAGCCAACUGCCUGGUCCAGACCAC 126% 13937 CDS 802 1946 CCAACUGCCUGGUCCAGACCACAGA 122% 13938 CDS 1166 1947 GUUCAUCAAGACCUGUGCCUGCCAU 106%

TABLE 13 Inhibition of gene expression with SPP1 sd-rxRNA sequences (Accession Number: NM_000582.2) % remaining Oligo Start SEQ ID SEQ ID expression (1 Number Site NO Sense sequence NO Antisense sequence uM A549) 14084 1025 1948 CUCAUGAAUUAGA 1949 UCUAAUUCAUGAGAA 61% AUAC 14085 1049 1950 CUGAGGUCAAUUA 1951 UAAUUGACCUCAGAA 50% GAUG 14086 1051 1952 GAGGUCAAUUAAA 1953 UUUAAUUGACCUCAG n/a AAGA 14087 1048 1954 UCUGAGGUCAAUU 1955 AAUUGACCUCAGAAG 69% AUGC 14088 1050 1956 UGAGGUCAAUUAA 1957 UUAAUUGACCUCAGA 76% AGAU 14089 1047 1958 UUCUGAGGUCAAU 1959 AUUGACCUCAGAAGA 60% UGCA 14090 800 1960 GUCAGCUGGAUGA 1961 UCAUCCAGCUGACUC 71% GUUU 14091 492 1962 UUCUGAUGAAUCU 1963 AGAUUCAUCAGAAUG n/a GUGA 14092 612 1964 UGGACUGAGGUCA 1965 UGACCUCAGUCCAUA n/a AACC 14093 481 1966 GAGUCUCACCAUU 1967 AAUGGUGAGACUCAU n/a CAGA 14094 614 1968 GACUGAGGUCAAA 1969 UUUGACCUCAGUCCA n/a UAAA 14095 951 1970 UCACAGCCAUGAA 1971 UUCAUGGCUGUGAAA 89% UUCA 14096 482 1972 AGUCUCACCAUUC 1973 GAAUGGUGAGACUCA 87% UCAG 14097 856 1974 AAGCGGAAAGCCA 1975 UGGCUUUCCGCUUAU 88% AUAA 14098 857 1976 AGCGGAAAGCCAA 1977 UUGGCUUUCCGCUUA 113% UAUA 14099 365 1978 ACCACAUGGAUGA 1979 UCAUCCAUGUGGUCA 98% UGGC 14100 359 1980 GCCAUGACCACAU 1981 AUGUGGUCAUGGCU 84% UUCGU 14101 357 1982 AAGCCAUGACCAC 1983 GUGGUCAUGGCUUU 88% CGUUG 14102 858 1984 GCGGAAAGCCAAU 1985 AUUGGCUUUCCGCUU n/a AUAU 14103 1012 1986 AAAUUUCGUAUUU 1987 AAAUACGAAAUUUCA 93% GGUG 14104 1014 1988 AUUUCGUAUUUCU 1989 AGAAAUACGAAAUUU 89% CAGG 14105 356 1990 AAAGCCAUGACCA 1991 UGGUCAUGGCUUUC 85% GUUGG 14106 368 1992 ACAUGGAUGAUAU 1993 AUAUCAUCCAUGUGG 67% UCAU 14107 1011 1994 GAAAUUUCGUAUU 1995 AAUACGAAAUUUCAG 87% GUGU 14108 754 1996 GCGCCUUCUGAUU 1997 AAUCAGAAGGCGCGU 73% UCAG 14109 1021 1998 AUUUCUCAUGAAU 1999 AUUCAUGAGAAAUAC 128% GAAA 14110 1330 2000 CUCUCAUGAAUAG 2001 CUAUUCAUGAGAGAA 101% UAAC 14111 346 2002 AAGUCCAACGAAA 2003 UUUCGUUGGACUUA 59% CUUGG 14112 869 2004 AUGAUGAGAGCAA 2005 UUGCUCUCAUCAUUG 89% GCUU 14113 701 2006 GCGAGGAGUUGAA 2007 UUCAACUCCUCGCUU 95% UCCA 14114 896 2008 UGAUUGAUAGUCA 2009 UGACUAUCAAUCACA 87% UCGG 14115 1035 2010 AGAUAGUGCAUCU 2011 AGAUGCACUAUCUAA 82% UUCA 14116 1170 2012 AUGUGUAUCUAUU 2013 AAUAGAUACACAUUC 36% AACC 14117 1282 2014 UUCUAUAGAAGAA 2015 UUCUUCUAUAGAAU 91% GAACA 14118 1537 2016 UUGUCCAGCAAUU 2017 AAUUGCUGGACAACC 152% GUGG 14119 692 2018 ACAUGGAAAGCGA 2019 UCGCUUUCCAUGUGU n/a GAGG 14120 840 2020 GCAGUCCAGAUUA 2021 UAAUCUGGACUGCUU 87% GUGG 14121 1163 2022 UGGUUGAAUGUGU 2023 ACACAUUCAACCAAU 31% AAAC 14122 789 2024 UUAUGAAACGAGU 2025 ACUCGUUUCAUAACU 96% GUCC 14123 841 2026 CAGUCCAGAUUAU 2027 AUAAUCUGGACUGCU 110% UGUG 14124 852 2028 AUAUAAGCGGAAA 2029 UUUCCGCUUAUAUAA 91% UCUG 14125 209 2030 UACCAGUUAAACA 2031 UGUUUAACUGGUAU 110% GGCAC 14126 1276 2032 UGUUCAUUCUAUA 2033 UAUAGAAUGAACAUA n/a GACA 14127 137 2034 CCGACCAAGGAAA 2035 UUUCCUUGGUCGGC 71% GUUUG 14128 711 2036 GAAUGGUGCAUAC 2037 GUAUGCACCAUUCAA 115% CUCC 14129 582 2038 AUAUGAUGGCCGA 2039 UCGGCCAUCAUAUGU 97% GUCU 14130 839 2040 AGCAGUCCAGAUU 2041 AAUCUGGACUGCUUG 102% UGGC 14131 1091 2042 GCAUUUAGUCAAA 2043 UUUGACUAAAUGCAA 10% AGUG 14132 884 2044 AGCAUUCCGAUGU 2045 ACAUCGGAAUGCUCA 93% UUGC 14133 903 2046 UAGUCAGGAACUU 2047 AAGUUCCUGACUAUC 97% AAUC 14134 1090 2048 UGCAUUUAGUCAA 2049 UUGACUAAAUGCAAA 39% GUGA 14135 474 2050 GUCUGAUGAGUCU 2051 AGACUCAUCAGACUG 99% GUGA 14136 575 2052 UAGACACAUAUGA 2053 UCAUAUGUGUCUACU 108% GUGG 14137 671 2054 CAGACGAGGACAU 2055 AUGUCCUCGUCUGUA 98% GCAU 14138 924 2056 CAGCCGUGAAUUC 2057 GAAUUCACGGCUGAC 100% UUUG 14139 1185 2058 AGUCUGGAAAUAA 2059 UUAUUUCCAGACUCA 47% AAUA 14140 1221 2060 AGUUUGUGGCUUC 2061 GAAGCCACAAACUAA 100% ACUA 14141 347 2062 AGUCCAACGAAAG 2063 CUUUCGUUGGACUU 103% ACUUG 14142 634 2064 AAGUUUCGCAGAC 2065 GUCUGCGAAACUUCU 100% UAGA 14143 877 2066 AGCAAUGAGCAUU 2067 AAUGCUCAUUGCUCU 104% CAUC 14144 1033 2068 UUAGAUAGUGCAU 2069 AUGCACUAUCUAAUU 95% CAUG 14145 714 2070 UGGUGCAUACAAG 2071 CUUGUAUGCACCAUU 101% CAAC 14146 791 2072 AUGAAACGAGUCA 2073 UGACUCGUUUCAUAA 100% CUGU 14147 813 2074 CCAGAGUGCUGAA 2075 UUCAGCACUCUGGUC 97% AUCC 14148 939 2076 CAGCCAUGAAUUU 2077 AAAUUCAUGGCUGUG 109% GAAU 14149 1161 2078 AUUGGUUGAAUGU 2079 ACAUUCAACCAAUAA 34% ACUG 14150 1164 2080 GGUUGAAUGUGUA 2081 UACACAUUCAACCAA n/a UAAA 14151 1190 2082 GGAAAUAACUAAU 2083 AUUAGUUAUUUCCA n/a GACUC 14152 1333 2084 UCAUGAAUAGAAA 2085 UUUCUAUUCAUGAG 31% AGAAU 14153 537 2086 GCCAGCAACCGAA 2087 UUCGGUUGCUGGCA n/a GGUCC 14154 684 2088 CACCUCACACAUG 2089 CAUGUGUGAGGUGA 100% UGUCC 14155 707 2090 AGUUGAAUGGUGC 2091 GCACCAUUCAACUCC 99% UCGC 14156 799 2092 AGUCAGCUGGAUG 2093 CAUCCAGCUGACUCG 95% UUUC 14157 853 2094 UAUAAGCGGAAAG 2095 CUUUCCGCUUAUAUA 106% AUCU 14158 888 2096 UUCCGAUGUGAUU 2097 AAUCACAUCGGAAUG 88% CUCA 14159 1194 2098 AUAACUAAUGUGU 2099 ACACAUUAGUUAUUU 95% CCAG 14160 1279 2100 UCAUUCUAUAGAA 2101 UUCUAUAGAAUGAAC 15% AUAG 14161 1300 2102 AACUAUCACUGUA 2103 UACAGUGAUAGUUU 86% GCAUU 14162 1510 2104 GUCAAUUGCUUAU 2105 AUAAGCAAUUGACAC 86% CACC 14163 1543 2106 AGCAAUUAAUAAA 2107 UUUAUUAAUUGCUG 110% GACAA 14164 434 2108 ACGACUCUGAUGA 2109 UCAUCAGAGUCGUUC 134% GAGU 14165 600 2110 UAGUGUGGUUUAU 2111 AUAAACCACACUAUC 102% ACCU 14166 863 2112 AAGCCAAUGAUGA 2113 UCAUCAUUGGCUUUC 93% CGCU 14167 902 2114 AUAGUCAGGAACU 2115 AGUUCCUGACUAUCA 101% AUCA 14168 921 2116 AGUCAGCCGUGAA 2117 UUCACGGCUGACUUU 98% GGAA 14169 154 2118 ACUACCAUGAGAA 2119 UUCUCAUGGUAGUG n/a AGUUU 14170 217 2120 AAACAGGCUGAUU 2121 AAUCAGCCUGUUUAA 66% CUGG 14171 816 2122 GAGUGCUGAAACC 2123 GGUUUCAGCACUCUG 102% GUCA 14172 882 2124 UGAGCAUUCCGAU 2125 AUCGGAAUGCUCAUU 103% GCUC 14173 932 2126 AAUUCCACAGCCA 2127 UGGCUGUGGAAUUC n/a ACGGC 14174 1509 2128 UGUCAAUUGCUUA 2129 UAAGCAAUUGACACC n/a ACCA 14175 157 2130 ACCAUGAGAAUUG 2131 CAAUUCUCAUGGUAG 109% UGAG 14176 350 2132 CCAACGAAAGCCA 2133 UGGCUUUCGUUGGA 95% CUUAC 14177 511 2134 CUGGUCACUGAUU 2135 AAUCAGUGACCAGUU 100% CAUC 14178 605 2136 UGGUUUAUGGACU 2137 AGUCCAUAAACCACA 99% CUAU 14179 811 2138 GACCAGAGUGCUG 2139 CAGCACUCUGGUCAU 88% CCAG 14180 892 2140 GAUGUGAUUGAUA 2141 UAUCAAUCACAUCGG 76% AAUG 14181 922 2142 GUCAGCCGUGAAU 2143 AUUCACGGCUGACUU 59% UGGA 14182 1169 2144 AAUGUGUAUCUAU 2145 AUAGAUACACAUUCA 69% ACCA 14183 1182 2146 UUGAGUCUGGAAA 2147 UUUCCAGACUCAAAU n/a AGAU 14184 1539 2148 GUCCAGCAAUUAA 2149 UUAAUUGCUGGACAA 77% CCGU 14185 1541 2150 CCAGCAAUUAAUA 2151 UAUUAAUUGCUGGA n/a CAACC 14186 427 2152 GACUCGAACGACU 2153 AGUCGUUCGAGUCAA 69% UGGA 14187 533 2154 ACCUGCCAGCAAC 2155 GUUGCUGGCAGGUCC 78% GUGG 18538 496 2156 GAUGAAUCUGAUA 2157 UAUCAGAUUCAUCAG 74% AAUG 18539 496 2158 UGAUGAAUCUGA 2159 UAUCAGAUUCAUCAG 72% UA AAUG 18540 175 2160 AUUUGCUUUUGCA 2161 UGCAAAAGCAAAUCA 98% CUGC 18541 175 2162 GAUUUGCUUUUG 2163 UGCAAAAGCAAAUCA 28% CA CUGC 18542 172 2164 GUGAUUUGCUUUA 2165 UAAAGCAAAUCACUG 24% CAAU 18543 172 2166 AGUGAUUUGCUU 2167 UAAAGCAAAUCACUG 14% UA CAAU 18544 1013 2168 AAUUUCGUAUUUA 2169 UAAAUACGAAAUUUC 100% AGGU 18545 1013 2170 AAAUUUCGUAUU 2171 UAAAUACGAAAUUUC 109% UA AGGU 18546 952 2172 CACAGCCAUGAAA 2173 UUUCAUGGCUGUGA 32% AAUUC 18547 952 2174 UCACAGCCAUGAAA 2175 UUUCAUGGCUGUGA 33% AAUUC 18548 174 2176 GAUUUGCUUUUGA 2177 UCAAAAGCAAAUCAC 57% UGCA 18549 174 2178 UGAUUUGCUUUU 2179 UCAAAAGCAAAUCAC 53% GA UGCA 18550 177 2180 UUGCUUUUGCCUA 2181 UAGGCAAAAGCAAAU 97% CACU 18551 177 2182 UUUGCUUUUGCC 2183 UAGGCAAAAGCAAAU 103% UA CACU 18552 1150 2184 UUUCUCAGUUUAA 2185 UUAAACUGAGAAAGA 96% AGCA 18553 1089 2186 UUGCAUUUAGUCA 2187 UGACUAAAUGCAAAG 94% UGAG 18554 1086 2188 ACUUUGCAUUUAA 2189 UUAAAUGCAAAGUGA n/a GAAA 18555 1093 2190 AUUUAGUCAAAAA 2191 UUUUUGACUAAAUG n/a CAAAG 18556 1147 2192 UUCUUUCUCAGUA 2193 UACUGAGAAAGAAGC n/a AUUU 18557 1148 2194 UCUUUCUCAGUUA 2195 UAACUGAGAAAGAAG 66% CAUU 18558 1128 2196 GAAAGAGAACAUA 2197 UAUGUUCUCUUUCA 16% UUUUG 18559 1087 2198 CUUUGCAUUUAGA 2199 UCUAAAUGCAAAGUG 28% AGAA 18560 1088 2200 UUUGCAUUUAGUA 2201 UACUAAAUGCAAAGU n/a GAGA 18561 1083 2202 CUCACUUUGCAUA 2203 UAUGCAAAGUGAGAA 53% AUUG 18562 1081 2204 UUCUCACUUUGCA 2205 UGCAAAGUGAGAAAU 89% UGUA 18563 555 2206 CACUCCAGUUGUA 2207 UACAACUGGAGUGAA 33% AACU 18564 1125 2208 AAUGAAAGAGAAA 2209 UUUCUCUUUCAUUU n/a UGCUA 18565 168 2210 UGCAGUGAUUUGA 2211 UCAAAUCACUGCAAU 14% UCUC 18566 1127 2212 UGAAAGAGAACAA 2213 UUGUUCUCUUUCAU 27% UUUGC 18567 1007 2214 ACCUGAAAUUUCA 2215 UGAAAUUUCAGGUG 129% UUUAU 18568 164 2216 GAAUUGCAGUGAA 2217 UUCACUGCAAUUCUC 47% AUGG 18569 222 2218 GGCUGAUUCUGGA 2219 UCCAGAAUCAGCCUG n/a UUUA

TABLE 14 Inhibition of gene expression with PTGS2 sd-rxRNA sequences (Accession Number: NM_000963.2) Oligo Start SEQ ID SEQ ID Number Site NO Sense sequence NO Antisense sequence % remaining expression (1 uM A549) 14422 451 2220 CACAUUUGAUUGA 2221 UCAAUCAAAUGUGAUC 72% UGG 14423 1769 2222 CACUGCCUCAAUU 2223 AAUUGAGGCAGUGUU 71% GAUG 14424 1464 2224 AAAUACCAGUCUU 2225 AAGACUGGUAUUUCAU 74% CUG 14425 453 2226 CAUUUGAUUGACA 2227 UGUCAAUCAAAUGUGA 83% UCU % remaining expression (1 uM PC-3) 17388 285 2228 GAAAACUGCUCAA 2229 UUGAGCAGUUUUCUCC 88% AUA 17389 520 2230 ACCUCUCCUAUUA 2231 UAAUAGGAGAGGUUA 25% GAGA 17390 467 2232 UCCACCAACUUAA 2233 UUAAGUUGGUGGACU 68% GUCA 17391 467 2234 GUCCACCAACUUAA 2235 UUAAGUUGGUGGACU 101% GUCA 17392 524 2236 CUCCUAUUAUACA 2237 UGUAUAAUAGGAGAG 49% GUUA 17393 448 2238 GAUCACAUUUGAA 2239 UUCAAAUGUGAUCUG 29% GAUG 17394 448 2240 AGAUCACAUUUGAA 2241 UUCAAAUGUGAUCUG 31% GAUG 17395 519 2242 AACCUCUCCUAUA 2243 UAUAGGAGAGGUUAG 12% AGAA 17396 437 2244 GUUGACAUCCAGA 2245 UCUGGAUGUCAACACA 86% UAA 17397 406 2246 CCUUCCUUCGAAA 2247 UUUCGAAGGAAGGGAA 23% UGU 17398 339 2248 ACUCCAAACACAA 2249 UUGUGUUUGGAGUGG 102% GUUU 17399 339 2250 CACUCCAAACACAA 2251 UUGUGUUUGGAGUGG 55% GUUU 17400 338 2252 CACUCCAAACACA 2253 UGUGUUUGGAGUGGG 62% UUUC 17401 468 2254 CCACCAACUUACA 2255 UGUAAGUUGGUGGAC 61% UGUC 17402 468 2256 UCCACCAACUUACA 2257 UGUAAGUUGGUGGAC 179% UGUC 17403 1465 2258 AAUACCAGUCUUA 2259 UAAGACUGGUAUUUCA 30% UCU 17404 243 2260 GACCAGUAUAAGA 2261 UCUUAUACUGGUCAAA 32% UCC 17405 1472 2262 GUCUUUUAAUGAA 2263 UUCAUUAAAAGACUGG 15% UAU 17406 2446 2264 AAUUUCAUGUCUA 2265 UAGACAUGAAAUUACU 142% GGU 17407 449 2266 AUCACAUUUGAUA 2267 UAUCAAAUGUGAUCUG 54% GAU 17408 449 2268 GAUCACAUUUGAUA 2269 UAUCAAAUGUGAUCUG 27% GAU 17409 444 2270 UCCAGAUCACAUA 2271 UAUGUGAUCUGGAUG 49% UCAA 17410 1093 2272 UACUGAUAGGAGA 2273 UCUCCUAUCAGUAUUA 32% GCC 17411 1134 2274 GUGCAACACUUGA 2275 UCAAGUGUUGCACAUA 70% AUC 17412 244 2276 ACCAGUAUAAGUA 2277 UACUUAUACUGGUCAA 63% AUC 17413 1946 2278 GAAGUCUAAUGAA 2279 UUCAUUAGACUUCUAC 19% AGU 17414 638 2280 AAGAAGAAAGUUA 2281 UAACUUUCUUCUUAGA 27% AGC 17415 450 2282 UCACAUUUGAUUA 2283 UAAUCAAAUGUGAUCU 216% GGA 17416 450 2284 AUCACAUUUGAUUA 2285 UAAUCAAAUGUGAUCU 32% GGA 17417 452 2286 ACAUUUGAUUGAA 2287 UUCAAUCAAAUGUGAU 99% CUG 17418 452 2288 CACAUUUGAUUGAA 2289 UUCAAUCAAAUGUGAU 54% CUG 17419 454 2290 AUUUGAUUGACAA 2291 UUGUCAAUCAAAUGUG 86% AUC 17420 454 2292 CAUUUGAUUGACAA 2293 UUGUCAAUCAAAUGUG 89% AUC 17421 1790 2294 CAUCUGCAAUAAA 2295 UUUAUUGCAGAUGAG 55% AGAC 17422 1790 2296 UCAUCUGCAAUAAA 2297 UUUAUUGCAGAUGAG 62% AGAC

TABLE 15 Inhibition of gene expression with CTGF sd-rxRNA sequences (Accession number: NM_001901.2) % remaining mRNA Oligo Start SEQ ID SEQ ID expression (1 uM Number Site NO Sense sequence NO Antisense sequence sd-rxRNA, A549) 13980 1222 2298 ACAGGAAGAUG 2299 UACAUCUUCCUGUAG 98% UA UACA 13981 813 2300 GAGUGGAGCGC 2301 AGGCGCUCCACUCUG 82% CU UGGU 13982 747 2302 CGACUGGAAGA 4206 UGUCUUCCAGUCGGU 116% CA AAGC 13983 817 2303 GGAGCGCCUGU 4207 GAACAGGCGCUCCAC 97% UC UCUG 13984 1174 2304 GCCAUUACAAC 4208 CAGUUGUAAUGGCAG 102% UG GCAC 13985 1005 2305 GAGCUUUCUG 4209 AGCCAGAAAGCUCAA 114% GCU ACUU 13986 814 2306 AGUGGAGCGCC 4210 CAGGCGCUCCACUCU 111% UG GUGG 13987 816 2307 UGGAGCGCCUG 4211 AACAGGCGCUCCACU 102% UU CUGU 13988 1001 2308 GUUUGAGCUU 4212 AGAAAGCUCAAACUU 99% UCU GAUA 13989 1173 2309 UGCCAUUACAA 4213 AGUUGUAAUGGCAG 107% CU GCACA 13990 749 2310 ACUGGAAGACA 4214 CGUGUCUUCCAGUCG 91% CG GUAA 13991 792 2311 AACUGCCUGGU 4215 GGACCAGGCAGUUGG 97% CC CUCU 13992 1162 2312 AGACCUGUGCC 4216 CAGGCACAGGUCUUG 107% UG AUGA 13993 811 2313 CAGAGUGGAGC 4217 GCGCUCCACUCUGUG 113% GC GUCU 13994 797 2314 CCUGGUCCAGA 4218 GGUCUGGACCAGGCA n/a CC GUUG 13995 1175 2315 CCAUUACAACU 4219 ACAGUUGUAAUGGCA 113% GU GGCA 13996 1172 2316 CUGCCAUUACA 4220 GUUGUAAUGGCAGG 110% AC CACAG 13997 1177 2317 AUUACAACUGU 4221 GGACAGUUGUAAUG 105% CC GCAGG 13998 1176 2318 CAUUACAACUG 4222 GACAGUUGUAAUGGC 89% UC AGGC 13999 812 2319 AGAGUGGAGCG 4223 GGCGCUCCACUCUGU 99% CC GGUC 14000 745 2320 ACCGACUGGAA 4224 UCUUCCAGUCGGUAA n/a GA GCCG 14001 1230 2321 AUGUACGGAGA 4225 UGUCUCCGUACAUCU 106% CA UCCU 14002 920 2322 GCCUUGCGAAG 4226 AGCUUCGCAAGGCCU 93% CU GACC 14003 679 2323 GCUGCGAGGAG 4227 CACUCCUCGCAGCAU 102% UG UUCC 14004 992 2324 GCCUAUCAAGU 4228 AAACUUGAUAGGCUU 100% UU GGAG 14005 1045 2325 AAUUCUGUGG 4229 ACUCCACAGAAUUUA 104% AGU GCUC 14006 1231 2326 UGUACGGAGAC 4230 AUGUCUCCGUACAUC 87% AU UUCC 14007 991 2327 AGCCUAUCAAG 4231 AACUUGAUAGGCUUG 101% UU GAGA 14008 998 2328 CAAGUUUGAGC 4232 AAGCUCAAACUUGAU 98% UU AGGC 14009 1049 2329 CUGUGGAGUA 4233 ACAUACUCCACAGAA 98% UGU UUUA 14010 1044 2330 AAAUUCUGUG 4234 CUCCACAGAAUUUAG 93% GAG CUCG 14011 1327 2331 UUUCAGUAGCA 4235 UGUGCUACUGAAAUC 95% CA AUUU 14012 1196 2332 CAAUGACAUCU 4236 AAAGAUGUCAUUGUC 101% UU UCCG 14013 562 2333 AGUACCAGUGC 4237 GUGCACUGGUACUUG 66% AC CAGC 14014 752 2334 GGAAGACACGU 4238 AAACGUGUCUUCCAG 95% UU UCGG 14015 994 2335 CUAUCAAGUUU 4239 UCAAACUUGAUAGGC 85% GA UUGG 14016 1040 2336 AGCUAAAUUCU 4240 ACAGAAUUUAGCUCG 61% GU GUAU 14017 1984 2337 AGGUAGAAUG 4241 UUACAUUCUACCUAU 32% UAA GGUG 14018 2195 2338 AGCUGAUCAGU 4242 AAACUGAUCAGCUAU 86% UU AUAG 14019 2043 2339 UUCUGCUCAGA 4243 UAUCUGAGCAGAAUU 81% UA UCCA 14020 1892 2340 UUAUCUAAGU 4244 UUAACUUAGAUAACU 84% UAA GUAC 14021 1567 2341 UAUACGAGUAA 4245 UAUUACUCGUAUAAG 72% UA AUGC 14022 1780 2342 GACUGGACAGC 4246 AAGCUGUCCAGUCUA 65% UU AUCG 14023 2162 2343 AUGGCCUUUAU 4247 UAAUAAAGGCCAUUU 80% UA GUUC 14024 1034 2344 AUACCGAGCUA 4248 UUUAGCUCGGUAUG 91% AA UCUUC 14025 2264 2345 UUGUUGAGAG 4249 ACACUCUCAACAAAU 58% UGU AAAC 14026 1032 2346 ACAUACCGAGC 4250 UAGCUCGGUAUGUC 106% UA UUCAU 14027 1535 2347 AGCAGAAAGGU 4251 UAACCUUUCUGCUGG 67% UA UACC 14028 1694 2348 AGUUGUUCCU 4252 UUAAGGAACAACUUG 94% UAA ACUC 14029 1588 2349 AUUUGAAGUG 4253 UUACACUUCAAAUAG 97% UAA CAGG 14030 928 2350 AAGCUGACCUG 4254 UCCAGGUCAGCUUCG 100% GA CAAG 14031 1133 2351 GGUCAUGAAGA 4255 CUUCUUCAUGACCUC 82% AG GCCG 14032 912 2352 AUGGUCAGGCC 4256 AAGGCCUGACCAUGC 84% UU ACAG 14033 753 2353 GAAGACACGUU 4257 CAAACGUGUCUUCCA 86% UG GUCG 14034 918 2354 AGGCCUUGCGA 4258 CUUCGCAAGGCCUGA 88% AG CCAU 14035 744 2355 UACCGACUGGA 4259 CUUCCAGUCGGUAAG 95% AG CCGC 14036 466 2356 ACCGCAAGAUC 4260 CCGAUCUUGCGGUUG 73% GG GCCG 14037 917 2357 CAGGCCUUGCG 4261 UUCGCAAGGCCUGAC 86% AA CAUG 14038 1038 2358 CGAGCUAAAUU 4262 AGAAUUUAGCUCGGU 84% CU AUGU 14039 1048 2359 UCUGUGGAGU 4263 CAUACUCCACAGAAU 87% AUG UUAG 14040 1235 2360 CGGAGACAUGG 4264 UGCCAUGUCUCCGUA 100% CA CAUC 14041 868 2361 AUGACAACGCC 4265 GAGGCGUUGUCAUU 104% UC GGUAA 14042 1131 2362 GAGGUCAUGAA 4266 UCUUCAUGACCUCGC 85% GA CGUC 14043 1043 2363 UAAAUUCUGU 4267 UCCACAGAAUUUAGC 74% GGA UCGG 14044 751 2364 UGGAAGACACG 4268 AACGUGUCUUCCAGU 84% UU CGGU 14045 1227 2365 AAGAUGUACGG 4269 CUCCGUACAUCUUCC 99% AG UGUA 14046 867 2366 AAUGACAACGC 4270 AGGCGUUGUCAUUG 94% CU GUAAC 14047 1128 2367 GGCGAGGUCAU 4271 UCAUGACCUCGCCGU 89% GA CAGG 14048 756 2368 GACACGUUUGG 4272 GGCCAAACGUGUCUU 93% CC CCAG 14049 1234 2369 ACGGAGACAUG 4273 GCCAUGUCUCCGUAC 100% GC AUCU 14050 916 2370 UCAGGCCUUGC 4274 UCGCAAGGCCUGACC 96% GA AUGC 14051 925 2371 GCGAAGCUGAC 4275 AGGUCAGCUUCGCAA 80% CU GGCC 14052 1225 2372 GGAAGAUGUAC 4276 CCGUACAUCUUCCUG 96% GG UAGU 14053 445 2373 GUGACUUCGGC 4277 GAGCCGAAGUCACAG 101% UC AAGA 14054 446 2374 UGACUUCGGCU 4278 GGAGCCGAAGUCACA 93% CC GAAG 14055 913 2375 UGGUCAGGCCU 4279 CAAGGCCUGACCAUG 67% UG CACA 14056 997 2376 UCAAGUUUGA 4280 AGCUCAAACUUGAUA 92% GCU GGCU 14057 277 2377 GCCAGAACUGC 4281 CUGCAGUUCUGGCCG 84% AG ACGG 14058 1052 2378 UGGAGUAUGU 4282 GGUACAUACUCCACA n/a ACC GAAU 14059 887 2379 GCUAGAGAAGC 4283 CUGCUUCUCUAGCCU 80% AG GCAG 14060 914 2380 GGUCAGGCCUU 4284 GCAAGGCCUGACCAU 112% GC GCAC 14061 1039 2381 GAGCUAAAUUC 4285 CAGAAUUUAGCUCGG 104% UG UAUG 14062 754 2382 AAGACACGUUU 4286 CCAAACGUGUCUUCC 109% GG AGUC 14063 1130 2383 CGAGGUCAUGA 4287 CUUCAUGACCUCGCC 103% AG GUCA 14064 919 2384 GGCCUUGCGAA 4288 GCUUCGCAAGGCCUG 109% GC ACCA 14065 922 2385 CUUGCGAAGCU 4289 UCAGCUUCGCAAGGC 106% GA CUGA 14066 746 2386 CCGACUGGAAG 4290 GUCUUCCAGUCGGUA 106% AC AGCC 14067 993 2387 CCUAUCAAGUU 4291 CAAACUUGAUAGGCU 67% UG UGGA 14068 825 2388 UGUUCCAAGAC 4292 AGGUCUUGGAACAGG 93% CU CGCU 14069 926 2389 CGAAGCUGACC 4293 CAGGUCAGCUUCGCA 95% UG AGGC 14070 923 2390 UUGCGAAGCUG 4294 GUCAGCUUCGCAAGG 95% AC CCUG 14071 866 2391 CAAUGACAACG 4295 GGCGUUGUCAUUGG 132% CC UAACC 14072 563 2392 GUACCAGUGCA 4296 CGUGCACUGGUACUU n/a CG GCAG 14073 823 2393 CCUGUUCCAAG 4297 GUCUUGGAACAGGCG 98% AC CUCC 14074 1233 2394 UACGGAGACAU 4298 CCAUGUCUCCGUACA 109% GG UCUU 14075 924 2395 UGCGAAGCUGA 4299 GGUCAGCUUCGCAAG 95% CC GCCU 14076 921 2396 CCUUGCGAAGC 4300 CAGCUUCGCAAGGCC 116% UG UGAC 14077 443 2397 CUGUGACUUCG 4301 GCCGAAGUCACAGAA 110% GC GAGG 14078 1041 2398 GCUAAAUUCUG 4302 CACAGAAUUUAGCUC 99% UG GGUA 14079 1042 2399 CUAAAUUCUGU 4303 CCACAGAAUUUAGCU 109% GG CGGU 14080 755 2400 AGACACGUUUG 4304 GCCAAACGUGUCUUC 121% GC CAGU 14081 467 2401 CCGCAAGAUCG 4305 GCCGAUCUUGCGGUU 132% GC GGCC 14082 995 2402 UAUCAAGUUU 4306 CUCAAACUUGAUAGG 105% GAG CUUG 14083 927 2403 GAAGCUGACCU 4307 CCAGGUCAGCUUCGC 114% GG AAGG 17356 1267 2404 ACAUUAACUCA 4308 UAUGAGUUAAUGUC 120% UA UCUCA 17357 1267 2405 GACAUUAACUC 2406 UAUGAGUUAAUGUC 56% AUA UCUCA 17358 1442 2407 UGAAGAAUGU 2408 UUAACAUUCUUCAAA 34% UAA CCAG 17359 1442 2409 UUGAAGAAUG 2410 UUAACAUUCUUCAAA 31% UUAA CCAG 17360 1557 2411 GAUAGCAUCUU 2412 UUAAGAUGCUAUCU 59% AA GAUGA 17361 1557 2413 AGAUAGCAUCU 2414 UUAAGAUGCUAUCU 47% UAA GAUGA 17362 1591 2415 UGAAGUGUAA 2416 UAAUUACACUUCAAA 120% UUA UAGC 17363 1599 2417 AAUUGAGAAGG 2418 UUCCUUCUCAAUUAC 71% AA ACUU 17364 1601 2419 UUGAGAAGGAA 2420 UUUUCCUUCUCAAUU 62% AA ACAC 17365 1732 2421 CAUUCUGAUUC 2422 UCGAAUCAGAAUGUC 99% GA AGAG 17366 1734 2423 UUCUGAUUCGA 2424 UUUCGAAUCAGAAUG 97% AA UCAG 17367 1770 2425 CUGUCGAUUAG 2426 UUCUAAUCGACAGGA 45% AA UUCC 17368 1805 2427 UUUGCCUGUAA 2428 UGUUACAGGCAAAUU 71% CA CACU 17369 1805 2429 AUUUGCCUGUA 2430 UGUUACAGGCAAAUU 67% ACA CACU 17370 1815 2431 ACAAGCCAGAU 2432 UAAUCUGGCUUGUU 65% UA ACAGG 17371 1815 2433 AACAAGCCAGA 2434 UAAUCUGGCUUGUU 35% UUA ACAGG 17372 2256 2435 CAGUUUAUUU 2436 UACAAAUAAACUGUC 113% GUA CGAA 17373 2265 2437 UGUUGAGAGU 2438 UACACUCUCAACAAA 35% GUA UAAA 17374 2265 2439 UUGUUGAGAG 2440 UACACUCUCAACAAA 31% UGUA UAAA 17375 2295 2441 UGCACCUUUCU 2442 UUAGAAAGGUGCAAA 34% AA CAUG 17376 2295 2443 UUGCACCUUUC 2444 UUAGAAAGGUGCAAA 28% UAA CAUG 17377 1003 2445 UUGAGCUUUC 2446 UCAGAAAGCUCAAAC 67% UGA UUGA 17378 2268 2447 UGAGAGUGUG 2448 UGUCACACUCUCAAC 42% ACA AAAU 17379 2272 2449 AGUGUGACCAA 2450 UUUUGGUCACACUCU 35% AA CAAC 17380 2272 2451 GAGUGUGACCA 2452 UUUUGGUCACACUCU 29% AAA CAAC 17381 2273 2453 GUGUGACCAAA 2454 UUUUUGGUCACACUC 42% AA UCAA 17382 2274 2455 UGUGACCAAAA 2456 UCUUUUGGUCACACU 42% GA CUCA 17383 2274 2457 GUGUGACCAAA 2458 UCUUUUGGUCACACU 37% AGA CUCA 17384 2275 2459 GUGACCAAAAG 2460 UACUUUUGGUCACAC 24% UA UCUC 17385 2277 2461 GACCAAAAGUU 2462 UUAACUUUUGGUCAC 27% AA ACUC 17386 2296 2463 GCACCUUUCUA 2464 UCUAGAAAGGUGCAA 23% GA ACAU 17387 2299 2465 CCUUUCUAGUU 2466 UCAACUAGAAAGGUG 46% GA CAAA

TABLE 16 Inhibition of gene expression with TGFB2 sd-rxRNA sequences (Accession Number: NM_001135599.1) % remaining Oligo Start SEQ ID SEQ ID expression Number Site NO Sense sequence NO Antisense sequence (1 uM, A549) 14408 1324 2467 GGCUCUCCUUCGA 2468 UCGAAGGAGAGCCAU 94% UCGC 14409 1374 2469 GACAGGAACCUGG 2470 CCAGGUUCCUGUCUU n/a UAUG 14410 946 2471 CCAAGGAGGUUUA 2472 UAAACCUCCUUGGCG 90% UAGU 14411 849 2473 AUUUCCAUCUACA 2474 UGUAGAUGGAAAUCA 72% CCUC 14412 852 2475 UCCAUCUACAACA 2476 UGUUGUAGAUGGAA 76% AUCAC 14413 850 2477 UUUCCAUCUACAA 2478 UUGUAGAUGGAAAU 98% CACCU 14414 944 2479 CGCCAAGGAGGUU 2480 AACCUCCUUGGCGUA 100% GUAC 14415 1513 2481 GUGGUGAUCAGAA 2482 UUCUGAUCACCACUG n/a GUAU 14416 1572 2483 CUCCUGCUAAUGU 2484 ACAUUAGCAGGAGAU 100% GUGG 14417 1497 2485 ACCUCCACAUAUA 2486 UAUAUGUGGAGGUG 73% CCAUC 14418 1533 2487 AAGUCCACUAGGA 2488 UCCUAGUGGACUUUA 98% UAGU 14419 1514 2489 UGGUGAUCAGAAA 2490 UUUCUGAUCACCACU 86% GGUA 14420 1534 2491 AGUCCACUAGGAA 2492 UUCCUAGUGGACUU 99% UAUAG 14421 943 2493 ACGCCAAGGAGGU 2494 ACCUCCUUGGCGUAG 41% UACU 18570 2445 2495 UAUUUAUUGUGUA 2496 UACACAAUAAAUAAC 79% UCAC 18571 2445 2497 UUAUUUAUUGUG 2498 UACACAAUAAAUAAC 75% UA UCAC 18572 2083 2499 AUCAGUGUUAAAA 2500 UUUUAACACUGAUGA 47% ACCA 18573 2083 2501 CAUCAGUGUUAAAA 2502 UUUUAACACUGAUGA 17% ACCA 18574 2544 2503 AUGGCUUAAGGAA 2504 UUCCUUAAGCCAUCC 59% AUGA 18575 2544 2505 GAUGGCUUAAGG 2506 UUCCUUAAGCCAUCC 141% AA AUGA 18576 2137 2507 UUGUGUUCUGUUA 2508 UAACAGAACACAAAC 77% UUCC 18577 2137 2509 UUUGUGUUCUGU 2510 UAACAGAACACAAAC 59% UA UUCC 18578 2520 2511 AAAUACUUUGCCA 2512 UGGCAAAGUAUUUG 75% GUCUC 18579 2520 2513 CAAAUACUUUGCCA 2514 UGGCAAAGUAUUUG 55% GUCUC 18580 3183 2515 CUUGCACUACAAA 2516 UUUGUAGUGCAAGU 84% CAAAC 18581 3183 2517 ACUUGCACUACAAA 2518 UUUGUAGUGCAAGU 80% CAAAC 18582 2267 2519 GAAUUUAUUAGUA 2520 UACUAAUAAAUUCUU 82% CCAG 18583 2267 2521 AGAAUUUAUUAG 2522 UACUAAUAAAUUCUU 67% UA CCAG 18584 3184 2523 UUGCACUACAAAA 2524 UUUUGUAGUGCAAG 77% UCAAA 18585 3184 2525 CUUGCACUACAAAA 2526 UUUUGUAGUGCAAG 59% UCAAA 18586 2493 2527 AUAAAACAGGUGA 2528 UCACCUGUUUUAUU 84% UUCCA 18587 2493 2529 AAUAAAACAGGUGA 2530 UCACCUGUUUUAUU 70% UUCCA 18588 2297 2531 GACAACAACAACA 2532 UGUUGUUGUUGUCG 40% UUGUU 18589 2046 2533 AUGCUUGUAACAA 2534 UUGUUACAAGCAUCA 39% UCGU 18590 2531 2535 CAGAAACUCAUGA 2536 UCAUGAGUUUCUGG 56% CAAAG 18591 2389 2537 GUAUUGCUAUGCA 2538 UGCAUAGCAAUACAG 64% AAAA 18592 2530 2539 CCAGAAACUCAUA 2540 UAUGAGUUUCUGGC 44% AAAGU 18593 2562 2541 ACUCAAACGAGCA 2542 UGCUCGUUUGAGUU 87% CAAGU 18594 2623 2543 AUAUGACCGAGAA 2544 UUCUCGGUCAUAUAA 69% UAAC 18595 2032 2545 CGACGACAACGAA 2546 UUCGUUGUCGUCGU 55% CAUCA 18596 2809 2547 GUAAACCAGUGAA 2548 UUCACUGGUUUACUA 58% AACU 18597 2798 2549 UUGUCAGUUUAGA 2550 UCUAAACUGACAAAG 38% AACC 18598 2081 2551 UCAUCAGUGUUAA 2552 UUAACACUGAUGAAC 25% CAAG 18599 2561 2553 AACUCAAACGAGA 2554 UCUCGUUUGAGUUC 57% AAGUU 18600 2296 2555 CGACAACAACAAA 2556 UUUGUUGUUGUCGU 69% UGUUC 18601 2034 2557 ACGACAACGAUGA 2558 UCAUCGUUGUCGUCG 22% UCAU 18602 2681 2559 GCUGCCUAAGGAA 2560 UUCCUUAGGCAGCUG 43% AUAC 18603 2190 2561 AUUCUACAUUUCA 2562 UGAAAUGUAGAAUAA 128% GGCC

TABLE 17 Inhibition of gene expression with TGFB1 sd-rxRNA sequences (Accession Number NM_000660.3) % remaining Oligo Start SEQ ID SEQ ID expression Number Site NO Sense sequence NO Antisense sequence (1 uM A549) 14394 1194 2563 GCUAAUGGUGGAA 2564 UUCCACCAUUAGCA 24% CGCGG 14395 2006 2565 UGAUCGUGCGCUC 2566 GAGCGCACGAUCAU 79% GUUGG 14396 1389 2567 CAAUUCCUGGCGA 2568 UCGCCAGGAAUUGU 77% UGCUG 14397 1787 2569 AGUGGAUCCACGA 2570 UCGUGGAUCCACUU n/a CCAGC 14398 1867 2571 UACAGCAAGGUCC 2572 GGACCUUGCUGUAC 82% UGCGU 14399 2002 2573 AACAUGAUCGUGC 2574 GCACGAUCAUGUUG n/a GACAG 14400 2003 2575 ACAUGAUCGUGCG 2576 CGCACGAUCAUGUU n/a GGACA 14401 1869 2577 CAGCAAGGUCCUG 2578 CAGGACCUUGCUGU 82% ACUGC 14402 2000 2579 CCAACAUGAUCGU 2580 ACGAUCAUGUUGGA 66% CAGCU 14403 986 2581 AGCGGAAGCGCAU 2582 AUGCGCUUCCGCUU 78% CACCA 14404 995 2583 GCAUCGAGGCCAU 2584 AUGGCCUCGAUGCG 79% CUUCC 14405 963 2585 GACUAUCGACAUG 2586 CAUGUCGAUAGUCU 80% UGCAG 14406 955 2587 ACCUGCAAGACUA 2588 UAGUCUUGCAGGUG 88% GAUAG 14407 1721 2589 GCUCCACGGAGAA 2590 UUCUCCGUGGAGCU n/a GAAGC 18454 1246 2591 CACAGCAUAUAUA 2592 UAUAUAUGCUGUG 58% UGUACU 18455 1248 2593 CAGCAUAUAUAUA 2594 UAUAUAUAUGCUGU 87% GUGUA 18456 1755 2595 GUACAUUGACUUA 2596 UAAGUCAAUGUACA 107% GCUGC 18457 1755 2597 UGUACAUUGACUUA 2598 UAAGUCAAUGUACA 77% GCUGC 18458 1708 2599 AACUAUUGCUUCA 2600 UGAAGCAAUAGUUG 75% GUGUC 18459 1708 2601 CAACUAUUGCUUCA 2602 UGAAGCAAUAGUUG 73% GUGUC 18460 1250 2603 GCAUAUAUAUGUA 2604 UACAUAUAUAUGCU n/a GUGUG 18461 1754 2605 UGUACAUUGACUA 2606 UAGUCAAUGUACAG 91% CUGCC 18462 1754 2607 CUGUACAUUGACUA 2608 UAGUCAAUGUACAG 92% CUGCC 18463 1249 2609 AGCAUAUAUAUGA 2610 UCAUAUAUAUGCUG n/a UGUGU 18464 1383 2611 CAGCAACAAUUCA 2612 UGAAUUGUUGCUG 77% UAUUUC 18465 1251 2613 CAUAUAUAUGUUA 2614 UAACAUAUAUAUGC 84% UGUGU 18466 1713 2615 UUGCUUCAGCUCA 2616 UGAGCUGAAGCAAU n/a AGUUG 18467 1713 2617 AUUGCUUCAGCUCA 2618 UGAGCUGAAGCAAU 83% AGUUG 18468 1247 2619 ACAGCAUAUAUAA 2620 UUAUAUAUGCUGU 96% GUGUAC 18469 1712 2621 AUUGCUUCAGCUA 2622 UAGCUGAAGCAAUA 90% GUUGG 18470 1712 2623 UAUUGCUUCAGCUA 2624 UAGCUGAAGCAAUA 98% GUUGG 18471 1212 2625 CAAGUUCAAGCAA 2626 UUGCUUGAACUUGU n/a CAUAG 18472 1222 2627 CAGAGUACACACA 2628 UGUGUGUACUCUGC 45% UUGAA 18473 1228 2629 ACACACAGCAUAA 2630 UUAUGCUGUGUGU 36% ACUCUG 18474 1233 2631 CAGCAUAUAUAUA 2632 UAUAUAUAUGCUGU 68% GUGUA 18475 1218 2633 UCAAGCAGAGUAA 2634 UUACUCUGCUUGAA 64% CUUGU 18476 1235 2635 AGCAUAUAUAUGA 2636 UCAUAUAUAUGCUG 78% UGUGU 18477 1225 2637 AGAGUACACACAA 2638 UUGUGUGUACUCU 92% GCUUGA 18478 1221 2639 AAGCAGAGUACAA 2640 UUGUACUCUGCUUG 103% AACUU 18479 1244 2641 UUCAACACAUCAA 2642 UUGAUGUGUUGAA 84% GAACAU 18480 1224 2643 AGCAGAGUACACA 2644 UGUGUACUCUGCUU 37% GAACU 18481 1242 2645 AUAUAUGUUCUUA 2646 UAAGAACAUAUAUA 62% UGCUG 18482 1213 2647 GACAAGUUCAAGA 2648 UCUUGAACUUGUCA 47% UAGAU 18483 1760 2649 UUAAAGAUGGAGA 2650 UCUCCAUCUUUAAU 69% GGGGC 18484 1211 2651 CUAUGACAAGUUA 2652 UAACUUGUCAUAGA n/a UUUCG 19411 1212 2653 CAACGAAAUCUAA 2654 UUAGAUUUCGUUG 52% UGGGUU 19412 1222 2655 UAUGACAAGUUCA 2656 UGAACUUGUCAUAG 51% AUUUC 19413 1228 2657 AAGUUCAAGCAGA 2658 UCUGCUUGAACUUG n/a UCAUA 19414 1233 2659 CAAGCAGAGUACA 2660 UGUACUCUGCUUGA 41% ACUUG 19415 1218 2661 AAUCUAUGACAAA 2662 UUUGUCAUAGAUU 104% UCGUUG 19416 1244 2663 CACACAGCAUAUA 2664 UAUAUGCUGUGUG 31% UACUCU

TABLE 18 Inhibition of gene expression with SPP1 sd-rxRNA sequences (Accession Number NM_000582.2) % remaining Oligo Start SEQ ID SEQ ID expression Number Site NO Sense sequence NO Antisense sequence (1 uM A549) 14084 1025 2665 mC.mU.mC. A.mU. 2666 P.mU.fC.fU. A. 61% G. A. A.mU.mU. A. A.fU.fU.fC. A.fU. G. A. G. A.Chl G* A* A* A*mU* A* C. 14085 1049 2667 mC.mU. G. A. G. 2668 P.mU. A. A.fU.fU. G. 50% G.mU.mC. A. A.fC.fC.fU.mC. A. G* A* A.mU.mU. A.Chl A* G* A*mU* G. 14086 1051 2669 G. A. G. G.mU.mC. 2670 P.mU.fU.fU. A. A.fU.fU. n/a A. A.mU.mU. A. A. G. A.fC.mC.mU.mC* A* A.Chl G* A* A* G* A. 14087 1048 2671 mU.mC.mU. G. A. G. 2672 P.mA. A.fU.fU. G. 69% G.mU.mC. A. A.fC.fC.fU.fC. A. G. A* A.mU.mU.Chl A* G* A*mU* G* C. 14088 1050 2673 mU. G. A. G. 2674 P.mU.fU. A. A.fU.fU. G. 76% G.mU.mC. A. A.fC.fC.mU.mC. A* G* A.mU.mU. A. A.Chl A* A* G* A* U. 14089 1047 2675 mU.mU.mC.mU. G. 2676 P.mA.fU.fU. G. 60% A. G. G.mU.mC. A. A.fC.fC.fU.fC. A. G. A. A.mU.Chl A* G* A*mU* G*mC* A. 14090 800 2677 G.mU.mC. A. 2678 P.mU.fC. A.fU.fC.fC. A. 71% G.mC.mU. G. G. G.fC.fU. G. A.mU. G. A.Chl A.mC*mU*mC* G*mU*mU* U. 14091 492 2679 mU.mU.mC.mU. G. 2680 P.mA. G. A.fU.fU.fC. n/a A.mU. G. A. A.fU.fC. A. G. A. A*mU* A.mU.mC.mU.Chl G* G*mU* G* A. 14092 612 2681 mU. G. G. A.mC.mU. 2682 P.mU. G. A.fC.fC.fU.fC. n/a G. A. G. G.mU.mC. A. G.fU.mC.mC. A*mU* A.Chl A* A* A*mC* C. 14093 481 2683 G. A. 2684 P.mA. A.fU. G. G.fU. G. n/a G.mU.mC.mU.mC. A. G. A.mC.mU.mC* A.mC.mC. A*mU*mC* A* G* A. A.mU.mU.Chl 14094 614 2685 G. A.mC.mU. G. A. 2686 P.mU.fU.fU. G. n/a G. G.mU.mC. A. A. A.fC.fC.fU.fC. A. A.Chl G.mU.mC*mC* A*mU* A* A* A. 14095 951 2687 mU.mC. A.mC. A. 2688 P.mU.fU.fC. A.fU. G. 89% G.mC.mC. A.mU. G. G.fC.fU. G.mU. G. A* A* A. A.Chl A*mU*mU*mC* A. 14096 482 2689 A. 2690 P.mG. A. A.fU. G. G.fU. 87% G.mU.mC.mU.mC. G. A. G. A.mC.mU*mC* A.mC.mC. A*mU*mC* A* G. A.mU.mU.mC.Chl 14097 856 2691 A. A. G.mC. G. G. A. 2692 P.mU. G. 88% A. A. G.mC.mC. A.Chl G.fC.fU.fU.fU.fC.fC. G.mC.mU.mU* A*mU* A*mU* A* A. 14098 857 2693 A. G.mC. G. G. A. A. 2694 P.mU.fU. G. 113% A. G.mC.mC. A. A.Chl G.fC.fU.fU.fU.fC.fC. G.mC.mU*mU* A*mU* A*mU* A. 14099 365 2695 A.mC.mC. A.mC. 2696 P.mU.fC. A.fU.fC.fC. 98% A.mU. G. G. A.mU. A.fU. G.fU. G. G. A.Chl G.mU*mC* A*mU* G* G* C. 14100 359 2697 G.mC.mC. A.mU. G. 2698 P.mA.fU. G.fU. G. 84% A.mC.mC. A.mC. G.fU.fC. A.fU. G. A.mU.Chl G.mC*mU*mU*mU*mC* G* U. 14101 357 2699 A. A. G.mC.mC. 2700 P.mG.fU. G. G.fU.fC. 88% A.mU. G. A.mC.mC. A.fU. G. A.mC.Chl G.mC.mU.mU*mU*mC* G*mU*mU* G. 14102 858 2701 G.mC. G. G. A. A. A. 2702 P.mA.fU.fU. G. n/a G.mC.mC. A. G.fC.fU.fU.fU.fC.mC. A.mU.Chl G.mC*mU*mU* A*mU* A* U. 14103 1012 2703 A. A. 2704 P.mA. A. A.fU. A.fC. G. 93% A.mU.mU.mU.mC. A. A. G.mU. A.mU.mU.mU*mC* A* A.mU.mU.mU.Chl G* G*mU* G. 14104 1014 2705 A.mU.mU.mU.mC. 2706 P.mA. G. A. A. A.fU. 89% G.mU. A.fC. G. A. A. A.mU.mU.mU.mC.mU. A.mU*mU*mU*mC* Chl A* G* G. 14105 356 2707 A. A. A. G.mC.mC. 2708 P.mU. G. G.fU.fC. A.fU. 85% A.mU. G. A.mC.mC. G. A.Chl G.fC.mU.mU.mU*mC* G*mU*mU* G* G. 14106 368 2709 A.mC. A.mU. G. G. 2710 P.mA.fU. A.fU.fC. 67% A.mU. G. A.mU. A.fU.fC.fC. A.mU. A.mU.Chl G.mU* G* G*mU*mC* A* U. 14107 1011 2711 G. A. A. 2712 P.mA. A.fU. A.fC. G. A. 87% A.mU.mU.mU.mC. A. A.fU.mU.mU.mC* A* G.mU. A.mU.mU.Chl G* G*mU* G* U. 14108 754 2713 G.mC. 2714 P.mA. A.fU.fC. A. G. A. 73% G.mC.mC.mU.mU.mC. A. G. G.mC. G.mC* mU. G. G*mU*mU*mC* A* G. A.mU.mU.Chl 14109 1021 2715 A.mU.mU.mU.mC.mU. 2716 P.mA.fU.fU.fC. A.fU. G. 128% mC. A.mU. G. A. A. G. A. A. A.mU* A.mU.Chl A*mC* G* A* A* A. 14110 1330 2717 mC.mU.mC.mU.mC. 2718 P.mC.fU. A.fU.fU.fC. 101% A.mU. G. A. A.mU. A. A.fU. G. A. G. A. G* A* G.Chl A*mU* A* A* C. 14111 346 2719 A. A. G.mU.mC.mC. 2720 P.mU.fU.fU.fC. G.fU.fU. 59% A. A.mC. G. A. A. G. G. A.mC.mU.mU* A.Chl A*mC*mU*mU* G* G. 14112 869 2721 A.mU. G. A.mU. G. 2722 P.mU.fU. 89% A. G. A. G.mC. A. G.fC.fU.fC.fU.fC. A.Chl A.fU.mC. A.mU*mU* G* G*mC*mU* U. 14113 701 2723 G.mC. G. A. G. G. A. 2724 P.mU.fU.fC. A. 95% G.mU.mU. G. A. A.fC.fU.fC.fC.fU.mC. A.Chl G.mC*mU*mU*mU*mC* mC* A. 14114 896 2725 mU. G. A.mU.mU. G. 2726 P.mU. G. A.fC.fU. 87% A.mU. A. G.mU.mC. A.fU.fC. A. A.mU.mC. A.Chl A*mC* A*mU*mC* G* G. 14115 1035 2727 A. G. A.mU. A. 2728 P.mA. G. A.fU. G.fC. 82% G.mU. G.mC. A.fC.fU. A.mU.mC.mU* A.mU.mC.mU.Chl A* A*mU*mU*mC* A. 14116 1170 2729 A.mU. G.mU. G.mU. 2730 P.mA. A.fU. A. G. A.fU. 36% A.mU.mC.mU. A.fC. A.mC. A.mU.mU.Chl A.mU*mU*mC* A* A*mC* C. 14117 1282 2731 mU.mU.mC.mU. 2732 P.mU.fU.fC.fU.fU.fC.fU. 91% A.mU. A. G. A. A. G. A.fU. A. G. A. A*mU* A. A.Chl G* A* A*mC* A. 14118 1537 2733 mU.mU. 2734 P.mA. A.fU.fU. G.fC.fU. 152% G.mU.mC.mC. A. G. G. A.mC. A. G.mC. A. A*mC*mC* G*mU* G* A.mU.mU.Chl G. 14119 692 2735 A.mC. A.mU. G. G. 2736 P.mU.fC. n/a A. A. A. G. C.mG. G.fC.fU.fU.fU.fC.fC. A.Chl A.mU. G.mU* G*mU* G* A* G* G. 14120 840 2737 G.mC. A. 2738 P.mU. A. A.fU.fC.fU. G. 87% G.mU.mC.mC. A. G. G. A.fC.mU. A.mU.mU. A.Chl G.mC*mU*mU* G*mU* G* G. 14121 1163 2739 mU. G. G.mU.mU. G. 2740 P.mA.fC. A.fC. 31% A. A.mU. G.mU. A.fU.fU.fC. A. A.mC.mC. G.mU.Chl A* A*mU* A* A* A* C. 14122 789 2741 mU.mU. A.mU. G. A. 2742 P.mA.fC.fU.fC. 96% A. A.mC. G. A. G.fU.fU.fU.fC. A.mU. A. G.mU.Chl A*mC*mU* G*mU*mC* C. 14123 841 2743 mC. A. 2744 P.mA.fU. A. A.fU.fC.fU. 110% G.mU.mC.mC. A. G. G. G. A.mC.mU. A.mU.mU. A.mU.Chl G*mC*mU*mU* G*mU* G. 14124 852 2745 A.mU. A.mU. A. A. 2746 P.mU.fU.fU.fC.fC. 91% G.mC. G. G. A. A. G.fC.fU.fU. A.mU. A.Chl A.mU* A* A*mU*mC*mU* G. 14125 209 2747 mU. A.mC.mC. A. 2748 P.mU. G.fU.fU.fU. A. 110% G.mU.mU. A. A. A.fC.fU. G. G.mU. A.mC. A.Chl A*mU* G* G*mC* A* C. 14126 1276 2749 mU. G.mU.mU.mC. 2750 P.mU. A.fU. A. G. A. n/a A.mU.mU.mC.mU. A.fU. G. A. A.mC. A.mU. A.Chl A*mU* A* G* A*mC* A. 14127 137 2751 mC.mC. G. A.mC.mC. 2752 P.mU.fU.fU.fC.fC.fU.fU. 71% A. A. G. G. A. A. G. G.fU.mC. G. G*mC* A.Chl G*mU*mU*mU* G. 14128 711 2753 G. A. A.mU. G. 2754 P.mG.fU. A.fU. G.fC. 115% G.mU. G.mC. A.mU. A.fC.fC. A.mU.mU.mC* A.mC.Chl A* A*mC*mU*mC* C. 14129 582 2755 A.mU. A.mU. G. 2756 P.mU.fC. G. G.fC.fC. 97% A.mU. G. G.mC.mC. A.fU.fC. A.mU. A.mU* G. A.Chl G*mU* G*mU*mC* U. 14130 839 2757 A. G.mC. A. 2758 P.mA. A.fU.fC.fU. G. G. 102% G.mU.mC.mC. A. G. A.fC.fU. G.mC.mU*mU* A.mU.mU.Chl G*mU* G* G* C. 14131 1091 2759 G.mC. 2760 P.mU.fU.fU. G. A.fC.fU. 10% A.mU.mU.mU. A. A. A. A.mU. G.mC* A* G.mU.mC. A. A. A* A* G*mU* G. A.Chl 14132 884 2761 A. G.mC. 2762 P.mA.fC. A.fU.fC. G. G. 93% A.mU.mU.mC.mC. G. A. A.fU. G.mC.mU*mC* A.mU. G.mU.Chl A*mU*mU* G* C. 14133 903 2763 mU. A. G.mU.mC. A. 2764 P.mA. A. 97% G. G. A. G.fU.fU.fC.fC.fU. G. A.mC.mU.mU.Chl A.mC.mU. A*mU*mC* A* A*mU* C. 14134 1090 2765 mU. G.mC. 2766 P.mU.fU. G. A.fC.fU. A. 39% A.mU.mU.mU. A. A. A.fU. G.mC. A* A* A* G.mU.mC. A. A.Chl G*mU* G* A. 14135 474 2767 G.mU.mC.mU. G. 2768 P.mA. G. A.fC.fU.fC. 99% A.mU. G. A. A.fU.fC. A. G. G.mU.mC.mU.Chl A.mC*mU* G* G*mU* G* A. 14136 575 2769 mU. A. G. A.mC. 2770 P.mU.fC. A.fU. A.fU. 108% A.mC. A.mU. A.mU. G.fU. G.fU.mC.mU. G. A.Chl A*mC*mU* G*mU* G* G. 14137 671 2771 mC. A. G. A.mC. G. 2772 P.mA.fU. 98% A. G. G. A.mC. G.fU.fC.fC.fU.fC. A.mU.Chl G.fU.mC.mU. G*mU* A* G*mC* A* U. 14138 924 2773 mC. A. G.mC.mC. 2774 P.mG. A. A.fU.fU.fC. 100% G.mU. G. A. A.fC. G. G.mC.mU. G* A.mU.mU.mC.Chl A*mC*mU*mU*mU* G. 14139 1185 2775 A. G.mU.mC.mU. G. 2776 P.mU.fU. 47% G. A. A. A.mU. A. A.fU.fU.fU.fC.fC. A. G. A.Chl A.mC.mU*mC* A* A* A*mU* A. 14140 1221 2777 A. G.mU.mU.mU. 2778 P.mG. A. A. G.fC.fC. 100% G.mU. G. A.fC. A. A. A.mC.mU* G.mC.mU.mU.mC.Chl A* A* A*mC*mU* A. 14141 347 2779 A. G.mU.mC.mC. A. 2780 P.mC.fU.fU.fU.fC. 103% A.mC. G. A. A. A. G.fU.fU. G. G. G.Chl A.mC.mU*mU* A*mC*mU*mU* G. 14142 634 2781 A. A. 2782 P.mG.fU.fC.fU. G.fC. G. 100% G.mU.mU.mU.mC. A. A. G.mC. A. G. A.mC.mU.mU*mC*mU* A.mC.Chl mU* A* G* A. 14143 877 2783 A. G.mC. A. A.mU. 2784 P.mA. A.fU. G.fC.fU.fC. 104% G. A. G.mC. A.fU.fU. A.mU.mU.Chl G.mC.mU*mC*mU*mC* A*mU* C. 14144 1033 2785 mU.mU. A. G. A.mU. 2786 P.mA.fU. G.fC. A.fC.fU. 95% A. G.mU. G.mC. A.fU.fC.mU. A. A.mU.Chl A*mU*mU*mC* A*mU* G. 14145 714 2787 mU. G. G.mU. G.mC. 2788 P.mC.fU.fU. G.fU. A.fU. 101% A.mU. A.mC. A. A. G.fC. A.mC.mC. G.Chl A*mU*mU*mC* A* A* C. 14146 791 2789 A.mU. G. A. A. 2790 P.mU. G. A.fC.fU.fC. 100% A.mC. G. A. G.fU.fU.fU.mC. A.mU* G.mU.mC. A.Chl A* A*mC*mU* G* U. 14147 813 2791 mC.mC. A. G. A. 2792 P.mU.fU.fC. A. G.fC. 97% G.mU. G.mC.mU. G. A.fC.fU.fC.mU. G. A. A.Chl G*mU*mC* A*mU*mC* C. 14148 939 2793 mC. A. G.mC.mC. 2794 P.mA. A. A.fU.fU.fC. 109% A.mU. G. A. A.fU. G. G.mC.mU. A.mU.mU.mU.Chl G*mU* G* G* A* A* U. 14149 1161 2795 A.mU.mU. G. 2796 P.mA.fC. A.fU.fU.fC. A. 34% G.mU.mU. G. A. A.fC.fC. A. A.mU* A* A* A.mU. G.mU.Chl A*mC*mU* G. 14150 1164 2797 G. G.mU.mU. G. A. 2798 P.m U. A.fC. A.fC. n/a A.mU. G.mU. G.mU. A.fU.fU.fC. A. A.Chl A.mC.mC* A* A*mU* A* A* A. 14151 1190 2799 G. G. A. A. A.mU. A. 2800 P.mA.fU.fU. A. G.fU.fU. n/a A.mC.mU. A. A.fU.fU.mU.mC.mC* A* A.mU.Chl G* A*mC*mU* C. 14152 1333 2801 mU.mC. A.mU. G. A. 2802 P.mU.fU.fU.fC.fU. 31% A.mU. A. G. A. A. A.fU.fU.fC. A.mU. G. A* A.Chl G* A* G* A* A* U. 14153 537 2803 G.mC.mC. A. G.mC. 2804 P.mU.fU.fC. G. G.fU.fU. n/a A. A.mC.mC. G. A. G.fC.fU. G. G.mC* A* A.Chl G* G*mU*mC* C. 14154 684 2805 mC. 2806 P.mC. A.fU. G.fU. G.fU. 100% A.mC.mC.mU.mC. G. A. G. G.mU. G* A.mC. A.mC. A.mU. A*mU* G*mU*mC* C. G.Chl 14155 707 2807 A. G.mU.mU. G. A. 2808 P.mG.fC. A.fC.fC. 99% A.mU. G. G.mU. A.fU.fU.fC. A. G.mC.Chl A.mC.mU*mC*mC*mU* mC* G* C. 14156 799 2809 A. G.mU.mC. A. 2810 P.mC. A.fU.fC.fC. A. 95% G.mC.mU. G. G. G.fC.fU. G. A.mU. G.Chl A.mC.mU*mC* G*mU*mU*mU* C. 14157 853 2811 mU. A.mU. A. A. 2812 P.mC.fU.fU.fU.fC.fC. 106% G.mC. G. G. A. A. A. G.fC.fU.fU. A.mU. G.Chl A*mU* A* A*mU*mC* U. 14158 888 2813 mU.mU.mC.mC. G. 2814 P.mA. A.fU.fC. A.fC. 88% A.mU. G.mU. G. A.fU.fC. G. G. A. A*mU* A.mU.mU.Chl G*mC*mU*mC* A. 14159 1194 2815 A.mU. A. A.mC.mU. 2816 P.mA.fC. A.fC. A.fU.fU. 95% A. A.mU. G.mU. A. G.fU.mU. G.mU.Chl A.mU*mU*mU*mC*mC* A* G. 14160 1279 2817 mU.mC. 2818 P.mU.fU.fC.fU. A.fU. A. 15% A.mU.mU.mC.mU. G. A. A.mU. G. A* A.mU. A. G. A. A.Chl A*mC* A*mU* A*G. 14161 1300 2819 A. A.mC.mU. 2820 P.mU. A.fC. A. G.fU. G. 86% A.mU.mC. A.mC.mU. A.fU. A. G.mU. A.Chl G.mU.mU*mU* G*mC* A*mU* U. 14162 1510 2821 G.mU.mC. A. 2822 P.mA.fU. A. A. G.fC. A. 86% A.mU.mU. A.fU.fU. G. A.mC* G.mC.mU.mU. A*mC*mC* A*mC* C. A.mU.Chl 14163 1543 2823 A. G.mC. A. 2824 P.mU.fU.fU. A.fU.fU. A. 110% A.mU.mU. A. A.mU. A.fU.fU. G.mC.mU* G* A. A. A.Chl G* A*mC* A* A. 14164 434 2825 A.mC. G. 2826 P.mU.fC. A.fU.fC. A. G. 134% A.mC.mU.mC.mU. G. A. G.fU.mC. A.mU. G. A.Chl G.mU*mU*mC* G* A* G* U. 14165 600 2827 mU. A. G.mU. G.mU. 2828 P.mA.fU. A. A. A.fC.fC. 102% G. G.mU.mU.mU. A.fC. A.mC.mU. A.mU.Chl A*mU*mC* A*mC*mC* U. 14166 863 2829 A. A. G.mC.mC. A. 2830 P.mU.fC. A.fU.fC. 93% A.mU. G. A.mU. G. A.fU.fU. G. A.Chl G.mC.mU.mU*mU*mC* mC* G*mC* U. 14167 902 2831 A.mU. A. G.mU.mC. 2832 P.mA. G.fU.fU.fC.fC.fU. 101% A. G. G. A. G.A.fC.mU. A.mU*mC* A.mC.mU.Chl A* A*mU*mC* A. 14168 921 2833 A. G.mU.mC. A. 2834 P.mU.fU.fC. A.fC. G. 98% G.mC.mC. G.mU. G. G.fC.fU. G. A. A.Chl A.mC.mU*mU*mU* G* G* A* A. 14169 154 2835 A.mC.mU. 2836 P.mU.fU.fC.fU.fC. A.fU. n/a A.mC.mC. A.mU. G. G. G.fU. A. G.mU* G* A. G. A. A.Chl A* G*mU*mU* U. 14170 217 2837 A. A. A.mC. A. G. 2838 P.mA. A.fU.fC. A. 66% G.mC.mU. G. G.fC.fC.fU. A.mU.mU.Chl G.mU.mU.mU* A* A*mC*mU* G* G. 14171 816 2839 G. A. G.mU. 2840 P.mG. G.fU.fU.fU.fC. A. 102% G.mC.mU. G. A. A. G.fC. A.mC.mC.Chl A.mC.mU.mC*mU* G* G*mU*mC* A. 14172 882 2841 mU. G. A. G.mC. 2842 P.mA.fU.fC. G. G. A. 103% A.mU.mU.mC.mC. G. A.fU. G.fC.mU.mC. A.mU.Chl A*mU*mU* G*mC*mU* C. 14173 932 2843 A. 2844 P.mU. G. G.fC.fU. G.fU. n/a A.mU.mU.mC.mC. G. G. A. A.mU.mU*mC* A.mC. A. G.mC.mC. A*mC* G* G* C. A.Chl 14174 1509 2845 mU. G.mU.mC. A. 2846 P.mU. A. A. G.fC. A. n/a A.mU.mU. A.fU.fU. G. A.mC. G.mC.mU.mU. A.Chl A*mC*mC* A*mC*mC* A. 14175 157 2847 A.mC.mC. A.mU. G. 2848 P.mC. A. 109% A. G. A. A.mU.mU. A.fU.fU.fC.fU.fC. A.fU. G.Chl G. G.mU* A* G*mU* G* A* G. 14176 350 2849 mC.mC. A. A.mC. G. 2850 P.mU. G. 95% A. A. A. G.mC.mC. G.fC.fU.fU.fU.fC. A.Chl G.fU.mU. G. G* A*mC*mU*mU* A* C. 14177 511 2851 mC.mU. G. 2852 P.mA. A.fU.fC. A. G.fU. 100% G.mU.mC. A.mC.mU. G. A.fC.mC. A. G. A.mU.mU.Chl G*mU*mU*mC* A*mU* C. 14178 605 2853 mU. G. 2854 P.mA. G.fU.fC.fC. A.fU. 99% G.mU.mU.mU. A. A. A.mC.mC. A*mC* A.mU. G. G. A*mC*mU* A* U. A.mC.mU.Chl 14179 811 2855 G. A.mC.mC. A. G. 2856 P.mC. A. G.fC. 88% A. G.mU. G.mC.mU. A.fC.fU.fC.fU. G. G.Chl G.mU.mC* A*mU*mC*mC* A* G. 14180 892 2857 G. A.mU. G.mU. G. 2858 P.mU. A.fU.fC. A. 76% A.mU.mU. G. A.mU. A.fU.fC. A.fC. A.Chl A.mU.mC* G* G* A* A*mU* G. 14181 922 2859 G.mU.mC. A. 2860 P.mA.fU.fU.fC. A.fC. G. 59% G.mC.mC. G.mU. G. G.fC.fU. G. A. A.mU.Chl A.mC*mU*mU*mU* G* G* A. 14182 1169 2861 A. A.mU. G.mU. 2862 P.mA.fU. A. G. A.fU. 69% G.mU. A.fC. A.fC. A.mU.mC.mU. A.mU.mU*mC* A* A.mU.Chl A*mC*mC* A. 14183 1182 2863 mU.mU. G. A. 2864 P.mU.fU.fU.fC.fC. A. G. n/a G.mU.mC.mU. G. G. A.fC.fU.mC. A. A* A. A. A.Chl A*mU* A* G* A* U. 14184 1539 2865 G.mU.mC.mC. A. 2866 P.mU.fU. A. A.fU.fU. 77% G.mC. A. A.mU.mU. G.fC.fU. G. G. A.mC* A* A. A.Chl A*mC*mC* G* U. 14185 1541 2867 mC.mC. A. G.mC. A. 2868 P.mU. A.fU.fU. A. n/a A.mU.mU. A. A.mU. A.fU.fU. G.fC.mU. G. G* A.Chl A*mC* A* A*mC*C. 14186 427 2869 G. A.mC.mU.mC. G. 2870 P.mA. G.fU.fC. 69% A. A.mC. G. G.fU.fU.fC. G. A. A.mC.mU.Chl G.mU.mC* A* A*mU* G* G* A. 14187 533 2871 A.mC.mC.mU. 2872 P.mG.fU.fU. G.fC.fU. G. 78% G.mC.mC. A. G.mC. G.fC. A. G. A. A.mC.Chl G.mU*mC*mC* G*mU* G* G. 18538 496 2873 G. A.mU. G. A. 2874 P.mU. A.fU.fC. A. G. 74% A.mU.mC.mU. G. A.fU.fU.fC. A.fU.fC* A* A.mU. A.Chl G* A* A*fU* G. 18539 496 2875 mU. G. A.mU. G. A. 2876 P.mU. A.fU.fC. A. G. 72% A.mU.mC.mU. G. A.fU.fU.fC. A.fU.fC* A* A.mU. A.Chl G* A* A*fU* G. 18540 175 2877 A.mU.mU.mU. 2878 P.mU. G.fC. A. A. A. A. 98% G.mC.mU.mU.mU.mU. G.fC. A. A. A.fU*fC* G.mC. A.Chl A*fC*fU*fG* C. 18541 175 2879 G. A.mU.mU.mU. 2880 P.mU. G.fC. A. A. A. A. 28% G.mC.mU.mU.mU.mU. G.fC. A. A. A.fU*fC* G.mC. A.Chl A*fC*fU*fG* C. 18542 172 2881 G.mU. G. 2882 P.mU. A. A. A. G.fC. A. 24% A.mU.mU.mU. A. A.fU.fC. A.fC*fU* G.mC.mU.mU.mU. G*fC* A* A* U. A.Chl 18543 172 2883 A. G.mU. G. 2884 P.mU. A. A. A. G.fC. A. 14% A.mU.mU.mU. A. A.fU.fC. A.fC*fU* G.mC.mU.mU.mU. G*fC* A* A* U. A.Chl 18544 1013 2885 A. 2886 P.mU. A. A. A.fU. A.fC. 100% A.mU.mU.mU.mC. G. A. A. A.fU.fU*fU*fC* G.mU. A* G* G* U. A.mU.mU.mU. A.Chl 18545 1013 2887 A. A. 2888 P.mU. A. A. A.fU. A.fC. 109% A.mU.mU.mU.mC. G. A. A. A.fU.fU*fU*fC* G.mU. A* G* G* U. A.mU.mU.mU. A.Chl 18546 952 2889 mC. A.mC. A. 2890 P.mU.fU.fU. C. A.fU. G. 32% G.mC.mC. A.mU. G. G.fC.fU. G.fU. G* A* A* A. A. A.Chl A*fU*fU* C. 18547 952 2891 mU.mC. A.mC. A. 2892 P.mU.fU.fU. C. A.fU. G. 33% G.mC.mC. A.mU. G. G.fC.fU. G.fU. G* A* A* A. A. A.Chl A*fU*fU* C. 18548 174 2893 G. A.mU.mU.mU. 2894 P.mU.fC. A. A. A. A. 57% G.mC.mU.mU.mU.mU. G.fC. A. A. A.fU.fC* G. A.Chl A*fC*fU* G*fC* A. 18549 174 2895 mU. G. 2896 P.mU.fC. A. A. A. A. 53% A.mU.mU.mU. G.fC. A. A. A.fU.fC* G.mC.mU.mU.mU.mU. A*fC*fU* G*fC* A. G. A.Chl 18550 177 2897 mU.mU. 2898 P.mU. A. G. G.fC. A. A. 97% G.mC.mU.mU.mU.mU. A. A. G.fC. A. A* G.mC.mC.mU. A*fU*fC* A*fC* U. A.Chl 18551 177 2899 mU.mU.mU. 2900 P.mU. A. G. G.fC. A. A. 103% G.mC.mU.mU.mU.mU. A. A. G.fC. A. A* G.mC.mC.mU. A*fU*fC* A*fC* U. A.Chl 18552 1150 2901 mU.mU.mU.mC.mU. 2902 P.mU.fU. A. A. A.fC.fU. 96% mC. A. G. A. G. A. A. A* G* A* G.mU.mU.mU. A. A* G*fC* A. A.Chl 18553 1089 2903 mU.mU. G.mC. 2904 P.mU. G. A.fC.fU. A. A. 94% A.mU.mU.mU. A. A.fU. G.fC. A. A* A* G.mU.mC. A.Chl G*fU* G* A* G. 18554 1086 2905 A.mC.mU.mU.mU. 2906 P.mU.fU. A. A. A.fU. n/a G.mC. G.fC. A. A. A. G.fU* G* A.mU.mU.mU. A. A* G* A* A* A. A.Chl 18555 1093 2907 A.mU.mU.mU. A. 2908 P.mU.fU.fU.fU.fU. G. n/a G.mU.mC. A. A. A. A. A.fC.fU. A. A. A.fU* A.Chl G*fC* A* A* A* G. 18556 1147 2909 mU.mU.mC.mU.mU. 2910 P.mU. A.fC.fU. G. A. G. n/a mU.mC.mU.mC. A. A. A. A. G. A. A* G*fC* G.mU. A.Chl A*fU*fU* U. 18557 1148 2911 mU.mC.mU.mU.mU. 2912 P.mU. A. A.fC.fU. G. A. 66% mC.mU.mC. A. G. A. A. A. G. A* A* G.mU.mU. A.Chl G*fC* A*fU* U. 18558 1128 2913 G. A. A. A. G. A. G. 2914 P.mU. A.fU. 16% A. A.mC. A.mU. G.fU.fU.fC.fU.fC.fU.fU.fU. A.Chl fC* A*fU*fU*fU*fU* G. 18559 1087 2915 mC.mU.mU.mU. 2916 P.mU.fC.fU. A. A. A.fU. 28% G.mC. G.fC. A. A. A. G*fU* G* A.mU.mU.mU. A. G. A* G* A* A. A.Chl 18560 1088 2917 mU.mU.mU. G.mC. 2918 P.mU. A.fC.fU. A. A. n/a A.mU.mU.mU. A. A.fU. G.fC. A. A. A* G.mU. A.Chl G*fU* G* A* G* A. 18561 1083 2919 mC.mU.mC. 2920 P.mU. A.fU. G.fC. A. A. 53% A.mC.mU.mU.mU. A. G.fU. G. A. G* A* A* G.mC. A.mU. A.Chl A*fU*fU* G. 18562 1081 2921 mU.mU.mC.mU.mC. 2922 P.mU. G.fC. A. A. A. 89% A.mC.mU.mU.mU. G.fU. G. A. G. A. A* G.mC. A.Chl A*fU*fU* G*fU* A. 18563 555 2923 mC. 2924 P.mU. A.fC. A. A.fC.fU. 33% A.mC.mU.mC.mC. A. G. G. A. G.fU. G* A* A* G.mU.mU. G.mU. A* A*fC*fU. A.Chl 18564 1125 2925 A. A.mU. G. A. A. A. 2926 P.mU.fU.fU.fC.fU.fC.fU. n/a G. A. G. A. A. A. Chl fU.fU.fC. A.fU.fU*fU*fU* G*fC*fU* A. 18565 168 2927 mU. G.mC. A. G.mU. 2928 P.mU.fC. A. A. A.fU.fC. 14% G. A.fC.fU. G.fC. A* A.mU.mU.mU.mG. A*fU*fU*fC*fU* C. A.Chl 18566 1127 2929 mU. G. A. A. A. G. A. 2930 P.mU.fU. 27% G. A. A.mC. A. A.Chl G.fU.fU.fC.fU.fC.fU.fU.fU. fC. A*fU*fU*fU*fU* G* C. 18567 1007 2931 A.mC.mC.mU. G. A. 2932 P.mU. G. A. A. 129% A. A.fU.fU.fU.fC. A. G. A.mU.mU.mU.mC. G.fU* G*fU*fU*fU* A* A.Chl U. 18568 164 2933 G. A. A.mU.mU. 2934 P.mU.fU.fC. A.fC.fU. 47% G.mC. A. G.mU. G. A. G.fC. A. A.Chl A.fU.fU.fC*fU*fC* A*fU* G* G. 18569 222 2935 G. G.mC.mU. G. 2936 P.mU.fC.fC. A. G. A. n/a A.mU.mU.mC.mU. A.fU.fC. A. G.fC.fC*fU* G. G. A.Chl G*fU*fU*fU* A. 20612 172 2937 A. G.mU. G. 2938 P.mU. A. A. A. G.fC. A. n/a A.mU.mU.mU. A. A.fU.mC. A.mC*mU* G.mC.mU.mU.mU. G*mC* A* A* U. A.Chl 20613 172 2939 A. G.mU. G. 2940 P.mU. A. A. A. G.fC. A. n/a A.mU.mU.mU. A. A.fU.fC. A.mC*fU* G.mC.mU.mU.mU. G*mC* A* A* U. A.Chl 20614 172 2941 A. G.mU. G. 2942 P.mU. A. A. A. G. C. A. 101% A.mU.mU.mU. A. A. U.mC. A.mC*mU* G.mC.mU.mU.mU. G*mC* A* A* U. A.Chl 20615 172 2943 A. G.mU. G. 2944 P.mU. A. A. A. G.fC. A. 104% A.mU.mU.mU. A. A.fU.mC. G.mC.mU.mU.mU. A.mC*mU*mG*mC*mA* A.Chl mA* U.

TABLE 19 Inhibition of gene expression with PTGS2 sd-rxRNA sequences (Accession Number: NM_000963.2) Oligo Start SEQ ID SEQ ID Number Site NO Sense sequence NO Antisense sequence % remaining expression (1 uM A549) 14422 451 2945 mC. A.mC. 2946 P.mU.fC. A. A.fU.fC. A. 72% A.mU.mU.mU. G. A. A.fU. G.mU. G* A.mU.mU. G. A.Chl A*mU*mC*mU* G* G. 14423 1769 2947 mC. A.mC.mU. 2948 P.mA. A.fU.fU. G. A. G. 71% G.mC.mC.mU.mC. A. G.fC. A. G.mU. A.mU.mU.Chl G*mU*mU* G* A*mU* G. 14424 1464 2949 A. A. A.mU. 2950 P.mA. A. G. A.fC.fU. G. 74% A.mC.mC. A. G.fU. G.mU.mC.mU.mU.Chl A.mU.mU.mU*mC* A*mU*mC*mU* G. 14425 453 2951 mC. A.mU.mU.mU. 2952 P.mU. G.fU.fC. A. 83% G. A.mU.mU. G. A.fU.fC. A. A. A.mU. A.mC. A.Chl G*mU* G* A*mU*mC* U. % remaining expression (1 uM PC-3) 17388 285 2953 G. A. A. A. 2954 P.mU.fU. G. A. G.fC. A. 88% A.mC.mU. G.fU.fU.fU.fU.fC*fU*fC* G.mC.mU.mC. A. fC* A*fU* A. A.Chl 17389 520 2955 A.mC.mC.mU.mC.mU. 2956 P.mU. A. A.fU. A. G. G. 25% mC.mC.mU. A. G. A. G. G.fU*fU* A* A.mU.mU. A.Chl G* A* G* A. 17390 467 2957 mU.mC.mC. 2958 P.mU.fU. A. A. G.fU.fU. 68% A.mC.mC. A. G. G.fU. G. G. A*fC*fU* A.mC.mU.mU. A. G*fU*fC* A. A.Chl % remaining expression (1 uM A549) 17391 467 2959 G.mU.mC.mC. 2960 P.mU.fU. A. A. G.fU.fU. 101% A.mC.mC. A. G. G.fU. G. G. A*fC*fU* A.mC.mU.mU. A. G*fU*fC* A. A.Chl 17392 524 2961 mC.mU.mC.mC.mU. 2962 P.mU. G.fU. A.fU. A. 49% A.mU.mU. A.mU. A.fU. A. G. G. A. G* A* A.mC. A.Chl G*G*fU*fU* A. 17393 448 2963 G. A.mU.mC. A.mC. 2964 P.mU.fU.fC. A. A. A.fU. 29% A.mU.mU.mU. G. A. G.fU. G. A.fU.fC*fU* G* A.Chl G* A*fU* G. 17394 448 2965 A. G. A.mU.mC. 2966 P.mU.fU.fC. A. A. A.fU. 31% A.mC. G.fU. G. A.fU.fC*fU* G* A.mU.mU.mU. G. A. G* A*fU* G. A.Chl 17395 519 2967 A. 2968 P.mU. A.fU. A. G. G. A. 12% A.mC.mC.mU.mC.mU. G.A. G. G.fU.fU* A* G* mC.mC.mU. A.mU. A* G* A* A. A.Chl 17396 437 2969 G.mU.mU. G. A.mC. 2970 P.mU.fC.fU. G. G. A.fU. 86% A.mU.mC.mC. A. G. G.fU.fC. A. A.fC* A*fC* A.Chl A*fU* A* A. 17397 406 2971 mC.mC.mU.mU.mC. 2972 P.mU.fU.fU.fC. G. A. A. 23% mC.mU.mU.mC. G. G. G. A. A. G. G* G* A* A. A. A.Chl A*fU* G* U. 17398 339 2973 A.mC.mU.mC.mC. 2974 P.mU.fU. G.fU. 102% A. A. A.mC. A.mC. A. G.fU.fU.fU. G. G. A. A.Chl G.fU* G* G* G*fU*fU* U. 17399 339 2975 mC. 2976 P.mU.fU. G.fU. 55% A.mC.mU.mC.mC. A. G.fU.fU.fU. G. G. A. A. A.mC. A.mC. A. G.fU* G* G* G*fU*fU* A.Chl U. 17400 338 2977 mC. 2978 P.mU. G.fU. G.fU.fU.fU. 62% A.mC.mU.mC.mC. A. G. G. A. G.fU. G* G* A. A.mC. A.mC. A.Chl G*fU*fU*fU* C. 17401 468 2979 mC.mC. A.mC.mC. A. 2980 P.mU. G.fU. A. A. 61% A.mC.mU.mU. A.mC. G.fU.fU. G. G.fU. G. G* A.Chl A*fC*fU* G*fU* C. 17402 468 2981 mU.mC.mC. 2982 P.mU. G.fU. A. A. 179% A.mC.mC. A. G.fU.fU. G. G.fU. G. G* A.mC.mU.mU. A.mC. A*fC*fU* G*fU* C. A.Chl 17403 1465 2983 A. A.mU. A.mC.mC. 2984 P.mU. A. A. G. A.fC.fU. 30% A. G. G.fU. A.fU.fU*fU*fC* G.mU.mC.mU.mU. A*fU*fC* U. A.Chl 17404 243 2985 G. A.mC.mC. A. 2986 P.mU.fC.fU.fU. A.fU. 32% G.mU. A.mU. A. A. A.fC.fU. G. G.fU.fC* A* G. A.Chl A* A*fU*fC* C. 17405 1472 2987 G.mU.mC.mU.mU.mU. 2988 P.mU.fU.fC. A.fU.fU. A. 15% mU. A. A.mU. G. A. A. A. G. A.fC*fU* G* A. A.Chl G*fU* A* U. 17406 2446 2989 A. 2990 P.mU. A. G. A.fC. A.fU. 142% A.mU.mU.mU.mC. G. A. A. A.fU.fU* A.mU. A*fC*fU* G* G* U. G.mU.mC.mU. A.Chl 17407 449 2991 A.mU.mC. A.mC. 2992 P.mU. A.fU.fC. A. A. 54% A.mU.mU.mU. G. A.fU. G.fU. G. A.mU. A.Chl A.fU*fC*fU* G* G* A* U. 17408 449 2993 G. A.mU.mC. A.mC. 2994 P.mU. A.fU.fC. A. A. 27% A.mU.mU.mU. G. A.fU. G.fU. G. A.mU. A.Chl A.fU*fC*fU* G* G* A* U. 17409 444 2995 mU.mC.mC. A. G. 2996 P.mU. A.fU. G.fU. G. 49% A.mU.mC. A.mC. A.fU.fC.fU. G. G. A*fU* A.mU. A.Chl G*fU*fC* A* A. 17410 1093 2997 mU. A.mC.mU. G. 2998 P.mU.fC.fU.fC.fC.fU. 32% A.mU. A. G. G. A. G. A.fU.fC. A. G.fU. A.Chl A*fU*fU* A* G*fC* C. 17411 1134 2999 G.mU. G.mC. A. 3000 P.mU.fC. A. A. G.fU. 70% A.mC. A.mC.mU.fU. G.fU.fU. G.mC. A.fC* G. A.Chl A*fU* A* A*fU* C. 17412 244 3001 A.mC.mC. A. G.mU. 3002 P.mU. A.fC.fU.fU. A.fU. 63% A.mU. A. A. G.mU. A.fC.fU. G. G.fU*fC* A* A.Chl A* A*fU* C. 17413 1946 3003 G. A. A. 3004 P.mU.fU.fC. A.fU.fU. A. 19% G.mU.mC.mU. A. G. A.fC.mU.fU.fC*fU* A.mU. G. A. A.Chl A*fC* A* G* U. 17414 638 3005 A. A. G. A. A. G. A. 3006 P.mU. A. 27% A. A. G.mU.mU. A.fC.fU.fU.fU.fC.fU.fU.fC. A.Chl fU.fU* A* G* A* A* G* C. 17415 450 3007 mU.mC. A.mC. 3008 P.mU. A. A.fU.fC. A. A. 216% A.mU.mU.mU. G. A.fU. G.fU. G. A.mU.mU. A.Chl A*fU*fC*fU* G* G* A. 17416 450 3009 A.mU.mC. A.mC. 3010 P.mU. A. A.fU.fC. A. A. 32% A.mU.mU.mU. G. A.fU. G.fU. G. A.mU.mU. A.Chl A*fU*fC*fU* G* G* A. 17417 452 3011 A.mC. 3012 P.mU.fU.fC. A. A.fU.fC. 99% A.mU.mU.mU. G. A. A. A.fU. G.fU* G* A.mU.mU. G. A. A*fU*fC*fU* G. A.Chl 17418 452 3013 mC. A.mC. 3014 P.mU.fU.fC. A. A.fU.fC. 54% A.mU.mU.mU. G. A. A. A.fU. G.fU* G* A.mU.mU. G. A. A*fU*fC*fU* G. A.Chl 17419 454 3015 A.mU.mU.mU. G. 3016 P.mU.fU. G.fU.fC. A. 86% A.mU.mU. G. A.mC. A.fU.fC. A. A. A.fU* A. A.Chl G*fU* G* A*fU* C. 17420 454 3017 mC. A.mU.mU.mU. 3018 P.mU.fU. G.fU.fC. A. 89% G. A.mU.mU. G. A.fU.fC. A. A. A.fU* A.mC. A. A.Chl G*fU* G* A*fU* C. 17421 1790 3019 mC. A.mU.mC.mU. 3020 P.mU.fU.fU. A.fU.fU. 55% G.mC. A. A.mU. A. A. G.fC. A. G. A.fU. G* A* A.Chl G* A* G* A* C. 17422 1790 3021 mU.mC. 3022 P.mU.fU.fU. A.fU.fU. 62% A.mU.mC.mU. G.mC. G.fC. A. G. A.fU. G* A* A. A.mU. A. A. A.Chl G* A* G* A* C. 21180 448 3023 G. A.mU.mC. A.mC. 3024 P.mU.fU.fC. A.mA. A.fU. 76% A.mU.mU.mU. G. A. G.fU. G. A.mU.mC*mU* A.TEG-Chl G* G* A*mU* G. 21181 448 3025 G. A.mU.mC. A.mC. 3026 P.mU.fU.fC. A.mA. A.fU. 37% A.mU.mU.mU. G. A. G.fU. G. A.TEG-Chl A.fU.fC*fU*mG*mG*mA* fU* G. 21182 448 3027 G. A.mU.mC. A.mC. 3028 P.mU.fU.fC. A. A. A.fU. 29% A.mU.mU.mU. G.fU. G. A.fU.fC*fU* G* G*mA*mA.TEG-Chl G* A*fU* G. 21183 448 3029 mG*mA*mU.mC. 3030 P.mU.fU.fC. A. A. A.fU. 46% A.mC. G.fU. G. A.fU.fC*fU* G* A.mU.mU.mU. G* A*fU* G. G*mA*mA.TEG-Chl 21184 448 3031 mG*mA*mU.mC.mA. 3032 P.mU.fU.fC. A. A. A.fU. 60% mC.mA.mU.mU.mU. G.fU. G. A.fU.fC*fU* G* mG*mA*mA.TEG- G* A*fU* G. Chl 21185 449 3033 G. A.mU.mC. A.mC. 3034 P.mU. A.fU.fC. A. A. 27% A.mU.mU.mU. G. A.fU. G.fU. G. A.mU. A.TEG-Chl A.fU.fC*fU* G* G* A*fU* G. 21186 449 3035 G. A.mU.mC. A.mC. 3036 P.mU. A.fU.fC. A. A. 57% A.mU.mU.mU. G. A.fU. G.fU. G. A.mU. A.TEG-Chl A.mU.mC*mU* G* G* A*mU* G. 21187 449 3037 G. A.mU.mC. A.mC. 3038 P.mU. A.fU.fC. A.mA. 54% A.mU.mU.mU. G. A.fU. G.fU. G. A.mU. A.TEG-Chl A.mU.mC*mU* G* G* A*mU* G. 21188 449 3039 G. A.mU.mC. A.mC. 3040 P.mU. A.fU.fC. A. A. 66% A.mU.mU.mU. G. A.fU. G.fU. G. A.mU. A.TEG-Chl A.mU.mC*mU*mG*mG* mA*mU* G. 21189 449 3041 G. A.mU.mC. A.mC. 3042 P.mU. A.fU.fC. A.mA. 44% A.mU.mU.mU. G. A.fU. G.fU. G. A.mU. A.TEG-Chl A.mU.mC*mU*mG*mG* mA*mU* G. 21190 449 3043 G. A.mU.mC. A.mC. 3044 P.mU. A.fU.fC. A. A. 52% A.mU.mU.mU. G. A.fU. G.fU. G. A.mU. A.TEG-Chl A.fU.fC*fU*mG*mG*mA* fU* G. 21191 449 3045 G. A.mU.mC. A.mC. 3046 P.mU. A.fU.fC. A.mA. 41% A.mU.mU.mU. G. A.fU. G.fU. G. A.mU. A.TEG-Chl A.fU.fC*fU*mG*mG*mA* fU* G. 21192 449 3047 G. A.mU.mC. A.mC. 3048 P.mU. A.fU.fC. A. A. 98% A.mU.mU.mU. G. A.fU. G.fU. G. A.mU. A.TEG-Chl A.fU.mC*fU*mG*mG* mA*fU* G. 21193 449 3049 G. A.mU.mC. A.mC. 3050 P.mU. A.fU.fC. A. A. 93% A.mU.mU.mU. G. A.fU. G.fU. G. A*mU*mA.TEG-Chl A.fU*fC*fU* G* G* A* U. 21194 449 3051 mG*mA*mU.mC. 3052 P.mU. A.fU.fC. A. A. 119% A.mC. A.fU. G.fU. G. A.mU.mU.mU. G. A.fU*fC*fU* G* G* A* A*mU*mA.TEG-Chl U. 21195 449 3053 mG*mA*mU.mC.mA. 3054 P.mU. A.fU.fC. A. A. 292% mC.mA.mU.mU.mU. A.fU. G.fU. G. mG.mA*mU*mA.TEG- A.fU*fC*fU* G* G* A* Chl U. 20620 449 3055 G. A.mU.mC. A.mC. 3056 P.mU. A.fU.fC. A. A. 24% A.mU.mU.mU. G. A.fU. G.fU. G. A.mU. A.Chl-TEG A.mU*mC*mU* G* G* A* U. 20621 449 3057 G. A.mU.mC. A.mC. 3058 P.mU. A.fU.fC. A. A. 5% A.mU.mU.mU. G. A.fU. G.fU. G. A.mU. A.Chl-TEG A.mU*fC*mU* G* G* A* U. 20622 449 3059 G. A.mU.mC. A.mC. 3060 P.mU. A. U. C. A. A. A. 25% A.mU.mU.mU. G. U. G. U. G. A.mU. A.Chl-TEG A.mU*mC*mU* G* G* A* U. 20623 449 3061 G. A.mU.mC. A.mC. 3062 P.mU. A.fU.fC. A. A. 14% A.mU.mU.mU. G. A.fU. G.fU. G. A.mU. A.Chl-TEG A.mU*mC*mU*mG*mG* mA* U. 20588 448 3063 G. A.mU.mC. A.mC. 3064 P.mU.fU.fC. A. A. A.fU. 17% A.mU.mU.mU. G. A. G.fU. G. A.mU.mC*mU* A.Chl-TEG G* G* A*mU* G. 20589 448 3065 G. A.mU.mC. A.mC. 3066 P.mU.fU.fC. A. A. A.fU. 40% A.mU.mU.mU. G. A. G.fU. G. A.mU.fC*mU* A.Chl-TEG G* G* A*fU* G. 20590 448 3067 G. A.mU.mC. A.mC. 3068 P.mU. U. C. A. A. A. U. 34% A.mU.mU.mU. G. A. G. U. G. A.mU.mC*mU* A.Chl-TEG G* G* A*mU* G. 20591 448 3069 G. A.mU.mC. A.mC. 3070 P.mU.fU.fC. A. A. A.fU. n/a A.mU.mU.mU. G. A. G.fU. G. A.Chl-TEG A.fU.fC*fU*mG*mG*mA* fU* G.

TABLE 20 Inhibition of gene expression with CTGF sd-rxRNA sequences (Accession Number: NM_001901.2) % remaining mRNA expression Oligo Start SEQ ID SEQ ID (1 uM sd-rxRNA, Number Site NO Sense sequence NO Antisense sequence A549) 13980 1222 3071 A.mC. A. G. G. A. 3072 P.mU. A.fC. 98% A. G. A.mU. G.mU. A.fU.fC.fU.fU.fC.fC.mU. A.Chl G.mU* A* G*mU* A*mC* A. 13981 813 3073 G. A. G.mU. G. G. 3074 P.mA. G. G.fC. 82% A. G.mC. G.fC.fU.fC.fC. G.mC.mC.mU.Chl A.mC.mU.mC*mU* G*mU* G* G* U. 13982 747 3075 mC. G. A.mC.mU. 3076 P.mU. 116% G. G. A. A. G. G.fU.fC.fU.fU.fC.fC. A. A.mC. A.Chl G.mU.mC. G* G*mU* A* A* G* C. 13983 817 3077 G. G. A. G.mC. 3078 P.mG. A. A.fC. A. G. 97% G.mC.mC.mU. G.fC. G.mU.mU.mC.Chl G.fC.mU.mC.mC* A*mC*mU*mC*mU* G. 13984 1174 3079 G.mC.mC. 3080 P.mC. A. G.fU.fU. 102% A.mU.mU. A.mC. G.fU. A. A.fU. G. A. A.mC.mU. G.mC* A* G* G*mC* G.Chl A* C. 13985 1005 3081 G. A. 3082 P.mA. G.fC.fC. A. G. A. 114% G.mC.mU.mU.mU. A. A. G.mC.mU.mC* mC.mU. G. A* A* A*mC*mU* U. G.mC.mU.Chl 13986 814 3083 A. G.mU. G. G. A. 3084 P.mC. A. G. G.fC. 111% G.mC. G.fC.fU.fC.fC. G.mC.mC.mU. A.mC.mU*mC*mU* G.Chl G*mU* G* G. 13987 816 3085 mU. G. G. A. 3086 P.mA. A.fC. A. G. G.fC. 102% G.mC. G.fC.fU.mC.mC. G.mC.mC.mU. A*mC*mU*mC*mU* G.mU.mU.Chl G* U. 13988 1001 3087 G.mU.mU.mU. G. 3088 P.mA. G. A. A. A. 99% A. G.fC.fU.fC. A. A. G.mC.mU.mU.mU. A.mC*mU*mU* G* mC.mU.Chl A*mU* A. 13989 1173 3089 mU. G.mC.mC. 3090 P.mA. G.fU.fU. G.fU. 107% A.mU.mU. A.mC. A. A.fU. G. G.mC. A* A. A.mC.mU.Chl G* G*mC* A*mC* A. 13990 749 3091 A.mC.mU. G. G. 3092 P.mC. G.fU. 91% A. A. G. A.mC. G.fU.fC.fU.fU.fC.fC. A. A.mC. G.Chl G.mU*mC* G* G*mU* A* A. 13991 792 3093 A. A.mC.mU. 3094 P.mG. G. A.fC.fC. A. G. 97% G.mC.mC.mU. G. G.fC. A. G.mU.mU* G* G.mU.mC.mC.Chl G*mC*mU*mC* U. 13992 1162 3095 A. G. 3096 P.mC. A. G. G.fC. A.fC. 107% A.mC.mC.mU. A. G. G.mU. G.mU.mC.mU*mU* G.mC.mC.mU. G* A*mU* G* A. G.Chl 13993 811 3097 mC. A. G. A. 3098 P.mG.fC. G.fC.fU.fC.fC. 113% G.mU. G. G. A. A.fC.fU.mC.mU. G.mC. G.mC.Chl G*mU* G* G*mU*mC* U. 13994 797 3099 mC.mC.mU. G. 3100 P.mG. G.fU.fC.fU. G. n/a G.mU.mC.mC. A. G. A.fC.fC. A. G. G. A.mC.mC.Chl G*mC* A* G*mU*mU* G. 13995 1175 3101 mC.mC. 3102 P.mA.fC. A. G.fU.fU. 113% A.mU.mU. A.mC. G.fU. A. A.mU. G. A. A.mC.mU. G*mC* A* G* G*mC* G.mU.Chl A. 13996 1172 3103 mC.mU. 3104 P.mG.fU.fU. G.fU. A. 110% G.mC.mC. A.fU. G. G.mC. A. G* A.mU.mU. A.mC. G*mC* A*mC* A* G. A. A.mC.Chl 13997 1177 3105 A.mU.mU. A.mC. 3106 P.mG. G. A.fC. A. 105% A. A.mC.mU. G.fU.fU. G.fU. A. G.mU.mC.mC.Chl A.mU* G* G*mC* A* G* G. 13998 1176 3107 mC. A.mU.mU. 3108 P.mG. A.fC. A. G.fU.fU. 89% A.mC. A. G.fU. A. A.mU. G* A.mC.mU. G*mC* A* G* G* C. G.mU.mC.Chl 13999 812 3109 A. G. A. G.mU. G. 3110 P.mG. G.fC. 99% G. A. G.mC. G.fC.fU.fC.fC. G.mC.mC.Chl A.fC.mU.mC.mU* G*mU* G* G*mU* C. 14000 745 3111 A.mC.mC. G. 3112 P.mU.fC.fU.fU.fC.fC. A. n/a A.mC.mU. G. G. A. G.fU.fC. G. G.mU* A* A. G. A.Chl A* G*mC*mC* G. 14001 1230 3113 A.mU. G.mU. 3114 P.mU. 106% A.mC. G. G. A. G. G.fU.fC.fU.fC.fC. G.fU. A.mC. A.Chl A.mC. A.mU*mC*mU*mU* mC*mC* U. 14002 920 3115 G.mC.mC.mU.mU. 3116 P.mA. G.fC.fU.fU.fC. 93% G.mC. G. A. A. G.fC. A. A. G. G.mC.mU.Chl G.mC*mC*mU* G* A*mC* C. 14003 679 3117 G.mC.mU. G.mC. 3118 P.mC. 102% G. A. G. G. A. A.fC.fU.fC.fC.fU.fC. G.mU. G.Chl G.fC. A. G.mC* A*mU*mU*mU*mC* C. 14004 992 3119 G.mC.mC.mU. 3120 P.mA. A. A.fC.fU.fU. G. 100% A.mU.mC. A. A. A.fU. A. G. G.mU.mU.mU.Chl G.mC*mU*mU* G* G* A* G. 14005 1045 3121 A. 3122 P.mA.fC.fU.fC.fC. A.fC. 104% A.mU.mU.mC.mU. A. G. A. G.mU. G. G. A. A.mU.mU*mU* A* G.mU.Chl G*mC*mU* C. 14006 1231 3123 mU. G.mU. A.mC. 3124 P.mA.fU. 87% G. G. A. G. A.mC. G.fU.fC.fU.fC.fC. G.fU. A.mU.Chl A.mC. A*mU*mC*mU*mU* mC* C. 14007 991 3125 A. G.mC.mC.mU. 3126 P.mA. A.fC.fU.fU. G. 101% A.mU.mC. A. A. A.fU. A. G. G.mU.mU.Chl G.mC.mU*mU* G* G* A* G* A. 14008 998 3127 mC. A. A. 3128 P.mA. A. G.fC.fU.fC. A. 98% G.mU.mU.mU. G. A. A.fC.mU.mU. G* A. A*mU* A* G* G* C. G.mC.mU.mU.Chl 14009 1049 3129 mC.mU. G.mU. G. 3130 P.mA.fC. A.fU. 98% G. A. G.mU. A.mU. A.fC.fU.fC.fC. A.mC. A. G.mU.Chl G* A* A*mU*mU*mU* A. 14010 1044 3131 A. A. 3132 P.mC.fU.fC.fC. A.fC. A. 93% A.mU.mU.mC.mU. G. A. A.mU.mU.mU* G.mU. G. G. A. A* G*mC*mU*mC* G. G.Chl 14011 1327 3133 mU.mU.mU.mC. 3134 P.mU. G.fU. G.fC.fU. 95% A. G.mU. A. G.mC. A.fC.fU. G. A. A. A.mC. A.Chl A*mU*mC* A*mU*mU* U. 14012 1196 3135 mC. A. A.mU. G. 3136 P.mA. A. A. G. A.fU. 101% A.mC. G.fU.fC. A.mU.mU. A.mU.mC.mU.mU. G*mU*mC*mU*mC* mU.Chl mC* G. 14013 562 3137 A. G.mU. 3138 P.mG.fU. G.fC. A.fC.fU. 66% A.mC.mC. A. G. G.fU. G.mU. G.mC. A.mC.mU*mU* A.mC.Chl G*mC* A* G* C. 14014 752 3139 G. G. A. A. G. 3140 P.mA. A. A.fC. G.fU. 95% A.mC. A.mC. G.fU.fC.fU.mU.mC.mC* G.mU.mU.mU.Chl A* G*mU*mC* G* G. 14015 994 3141 mC.mU. 3142 P.mU.fC. A. A. 85% A.mU.mC. A. A. A.fC.fU.fU. G. A.mU. G.mU.mU.mU. G. A. G* A.Chl G*mC*mU*mU* G* G. 14016 1040 3143 A. G.mC.mU. A. 3144 P.mA.fC. A. G. A. 61% A. A.fU.fU.fU. A. A.mU.mU.mC.mU. G.mC.mU*mC* G* G.mU.Chl G*mU* A* U. 14017 1984 3145 A. G. G.mU. A. G. 3146 P.mU.fU. A.fC. 32% A. A.mU. G.mU. A. A.fU.fU.fC.fU. A.Chl A.mC.mC.mU* A*mU* G* G*mU* G. 14018 2195 3147 A. G.mC.mU. G. 3148 P.mA. A. A.fC.fU. G. 86% A.mU.mC. A. A.fU.fC. A. G.mC.mU* G.mU.mU.mU.Chl A*mU* A*mU* A* G. 14019 2043 3149 mU.mU.mC.mU. 3150 P.mU. A.fU.fC.fU. G. A. 81% G.mC.mU.mC. A. G.fC. A. G. A. G. A.mU. A.Chl A*mU*mU*mU*mC* mC* A. 14020 1892 3151 mU.mU. 3152 P.mU.fU. A. 84% A.mU.mC.mU. A. A.fC.fU.fU. A. G. A. G.mU.mU. A. A.mU. A. A*mC*mU* A.Chl G*mU* A* C. 14021 1567 3153 mU. A.mU. A.mC. 3154 P.mU. A.fU.fU. 72% G. A. G.mU. A. A.fC.fU.fC. G.fU. A.mU. A.Chl A.mU. A* A* G* A*mU* G* C. 14022 1780 3155 G. A.mC.mU. G. 3156 P.mA. A. G.fC.fU. 65% G. A.mC. A. G.fU.fC.fC. A. G.mC.mU.mU.Chl G.mU.mC*mU* A* A*mU*mC* G. 14023 2162 3157 A.mU. G. 3158 P.mU. A. A.fU. A. A. A. 80% G.mC.mC.mU.mU. G. G.fC.mC. mU. A.mU.mU. A.mU*mU*mU* A.Chl G*mU*mU* C. 14024 1034 3159 A.mU. A.mC.mC. 3160 P.mU.fU.fU. A. 91% G. A. G.mC.mU. A. G.fC.fU.fC. G. G.mU. A. A.Chl A.mU* G*mU*mC*mU*mU* C. 14025 2264 3161 mU.mU. 3162 P.mA.fC. 58% G.mU.mU. G. A. A.fC.fU.fC.fU.fC. A. G. A. G.mU. A.mC. A. A* A*mU* G.mU.Chl A* A* A* C. 14026 1032 3163 A.mC. A.mU. 3164 P.mU. A. G.fC.fU.fC. G. 106% A.mC.mC. G. A. G.fU. A.mU. G.mC.mU. A.Chl G.mU*mC*mU*mU* mC* A* U. 14027 1535 3165 A. G.mC. A. G. A. 3166 P.mU. A. 67% A. A. G. G.mU.mU. A.fC.fC.fU.fU.fU.fC.fU. A.Chl G.mC.mU* G* G*mU* A*mC* C. 14028 1694 3167 A. G.mU.mU. 3168 P.mU.fU. A. A. G. G. A. 94% G.mU.mU.mC.mC. A.fC. A. mU.mU. A. A.Chl A.mC.mU*mU* G* A*mC*mU* C. 14029 1588 3169 A.mU.mU.mU. G. 3170 P.mU.fU. A.fC. 97% A. A. G.mU. G.mU. A.fC.fU.fU.fC. A. A. A. A.Chl A.mU* A* G*mC* A* G* G. 14030 928 3171 A. A. G.mC.mU. 3172 P.mU.fC.fC. A. G. 100% G. A.mC.mC.mU. G.fU.fC. A. G. G. A.Chl G.mC.mU.mU*mC* G*mC* A* A* G. 14031 1133 3173 G. G.mU.mC. 3174 P.mC.fU.fU.fC.fU.fU.fC. 82% A.mU. G. A. A. G. A.fU. G. A. A. G.Chl A.mC.mC*mU*mC* G*mC*mC* G. 14032 912 3175 A.mU. G. 3176 P.mA. A. G. G.fC.fC.fU. 84% G.mU.mC. A. G. G. A.fC.mC. A.mU* G.mC.mC.mU.mU. G*mC* A*mC* A* G. Chl 14033 753 3177 G. A. A. G. A.mC. 3178 P.mC. A. A. A.fC. G.fU. 86% A.mC. G.fU.fC.mU.mU.mC*mC* G.mU.mU.mU. A* G*mU*mC* G. G.Chl 14034 918 3179 A. G. 3180 P.mC.fU.fU.fC. G.fC. A. 88% G.mC.mC.mU.mU. A. G. G.mC.mC.mU* G.mC. G. A. A. G* A*mC*mC* A* U. G.Chl 14035 744 3181 mU. A.mC.mC. G. 3182 P.mC.fU.fU.fC.fC. A. 95% A.mC.mU. G. G. A. G.fU.fC. G. G.mU. A* A. G.Chl A* G*mC*mC* G* C. 14036 466 3183 A.mC.mC. G.mC. 3184 P.mC.fC. G. 73% A. A. G. A.mU.mC. A.fU.fC.fU.fU. G.fC. G. G. G.Chl G.mU*mU* G* G*mC*mC* G. 14037 917 3185 mC. A. G. 3186 P.mU.fU.fC. G.fC. A. A. 86% G.mC.mC.mU.mU. G. G.fC.mC.mU. G* G.mC. G. A. A.Chl A*mC*mC* A*mU* G. 14038 1038 3187 mC. G. A. 3188 P.mA. G. A. 84% G.mC.mU. A. A. A.fU.fU.fU. A. A.mU.mU.mC.mU. G.fC.mU.mC. G* Chl G*mU* A*mU* G* U. 14039 1048 3189 mU.mC.mU. 3190 P.mC. A.fU. 87% G.mU. G. G. A. A.fC.fU.fC.fC. A.fC. A. G.mU. A.mU. G. A* G.Chl A*mU*mU*mU* A* G. 14040 1235 3191 mC. G. G. A. G. 3192 P.mU. G.fC.fC. A.fU. 100% A.mC. A.mU. G. G.fU.fC.fU.mC.mC. G.mC. A.Chl G*mU* A*mC* A*mU* C. 14041 868 3193 A.mU. G. A.mC. 3194 P.mG. A. G. G.fC. 104% A. A.mC. G.fU.fU. G.fU.mC. G.mC.mC.mU.mC. A.mU*mU* G* Chl G*mU* A* A. 14042 1131 3195 G. A. G. 3196 P.mU.fC.fU.fU.fC. 85% G.mU.mC. A.mU. A.fU. G. G. A. A. G. A.Chl A.fC.mC.mU.mC* G*mC*mC* G*mU* C. 14043 1043 3197 mU. A. A. 3198 P.mU.fC.fC. A.fC. A. G. 74% A.mU.mU.mC.mU. A. A.fU.mU.mU. A* G.mU. G. G. A.Chl G*mC*mU*mC* G* G. 14044 751 3199 mU. G. G. A. A. G. 3200 P.mA. A.fC. G.fU. 84% A.mC. A.mC. G.fU.fC.fU.fU.mC.mC. G.mU.mU.Chl A* G*mU*mC* G* G* U. 14045 1227 3201 A. A. G. A.mU. 3202 P.mC.fU.fC.fC. G.fU. 99% G.mU. A.mC. G. G. A.fC. A. G.Chl A.fU.mC.mU.mU*mC* mC*mU* G*mU* A. 14046 867 3203 A. A.mU. G. 3204 P.mA. G. G.fC. G.fU.fU. 94% A.mC. A. A.mC. G.fU.fC. A.mU.mU* G* G.mC.mC.mU.Chl G*mU* A* A* C. 14047 1128 3205 G. G.mC. G. A. G. 3206 P.mU.fC. A.fU. G. 89% G.mU.mC. A.mU. A.fC.fC.fU.fC. G. A.Chl G.mC.mC* G*mU*mC* A* G* G. 14048 756 3207 G. A.mC. A.mC. 3208 P.mG. G.fC.fC. A. A. 93% G.mU.mU.mU. G. A.fC. G.fU. G.mC.mC.Chl G.mU.mC*mU*mU*mC* mC* A* G. 14049 1234 3209 A.mC. G. G. A. G. 3210 P.mG.fC.fC. A.fU. 100% A.mC. A.mU. G. G.fU.fC.fU.fC.mC. G.mC.Chl G.mU* A*mC* A*mU*mC* U. 14050 916 3211 mU.mC. A. G. 3212 P.mU.fC. G.fC. A. A. G. 96% G.mC.mC.mU.mU. G.fC.fC.mU. G. G.mC. G. A.Chl A*mC*mC* A*mU* G* C. 14051 925 3213 G.mC. G. A. A. 3214 P.mA. G. G.fU.fC. A. 80% G.mC.mU. G. G.fC.fU.fU.mC. G.mC* A.mC.mC.mU.Chl A* A* G* G*mC* C. 14052 1225 3215 G. G. A. A. G. 3216 P.mC.fC. G.fU. A.fC. 96% A.mU. G.mU. A.fU.fC.fU.mU.mC.mC* A.mC. G. G.Chl mU* G*mU* A* G* U. 14053 445 3217 G.mU. G. 3218 P.mG. A. G.fC.fC. G. A. 101% A.mC.mU.mU.mC. A. G.fU.mC. A.mC* A* G. G* A* A* G* A. G.mC.mU.mC.Chl 14054 446 3219 mU. G. 3220 P.mG. G. A. G.fC.fC. G. 93% A.mC.mU.mU.mC. A. A. G.mU.mC. G. A*mC* A* G* A* A* G.mC.mU.mC.mC. G. Chl 14055 913 3221 mU. G. G.mU.mC. 3222 P.mC. A. A. G. 67% A. G. G.fC.fC.fU. G. G.mC.mC.mU.mU. A.mC.mC. A*mU* G.Chl G*mC* A*mC* A. 14056 997 3223 mU.mC. A. A. 3224 P.mA. G.fC.fU.fC. A. A. 92% G.mU.mU.mU. G. A.fC.fU.mU. G. A*mU* A. G.mC.mU.Chl A* G* G*mC* U. 14057 277 3225 G.mC.mC. A. G. 3226 P.mC.fU. G.fC. A. 84% A. A.mC.mU. G.fU.fU.fC.fU. G. G.mC. A. G.Chl G.mC*mC* G* A*mC* G* G. 14058 1052 3227 mU. G. G. A. 3228 P.mG. G.fU. A.fC. A.fU. n/a G.mU. A.mU. A.fC.fU.mC.mC. G.mU. A*mC* A* G* A* A* A.mC.mC.Chl U. 14059 887 3229 G.mC.mU. A. G. 3230 P.mC.fU. 80% A. G. A. A. G.mC. G.fC.fU.fU.fC.fU.fC.fU. A. G.Chl A. G.mC*mC*mU* G*mC* A* G. 14060 914 3231 G. G.mU.mC. A. 3232 P.mG.fC. A. A. G. 112% G. G.fC.fC.fU. G. G.mC.mC.mU.mU. A.mC.mC* A*mU* G.mC.Chl G*mC* A* C. 14061 1039 3233 G. A. G.mC.mU. 3234 P.mC. A. G. A. 104% A. A. A.fU.fU.fU. A. A.mU.mU.mC.mU. G.mC.mU.mC* G* G.Chl G*mU* A*mU* G. 14062 754 3235 A. A. G. A.mC. 3236 P.mC.fC. A. A. A.fC. 109% A.mC. G.fU. G.mU.mU.mU. G. G.fU.mC.mU.mU*mC* G.Chl mC* A* G*mU* C. 14063 1130 3237 mC. G. A. G. 3238 P.mC.fU.fU.fC. A.fU. G. 103% G.mU.mC. A.mU. A.fC.fC.mU.mC. G. A. A. G.Chl G*mC*mC* G*mU*mC* A. 14064 919 3239 G. 3240 P.mG.fC.fU.fU.fC. 109% G.mC.mC.mU.mU. G.fC. A. A. G. G.mC. G. A. A. G.mC.mC*mU* G* G.mC.Chl A*mC*mC* A. 14065 922 3241 mC.mU.mU. 3242 P.mU.fC. A. 106% G.mC. G. A. A. G.fC.fU.fU.fC. G.fC. A. G.mC.mU. G. A. G* G*mC*mC*mU* A.Chl G* A. 14066 746 3243 mC.mC. G. 3244 P.mG.fU.fC.fU.fU.fC.fC. 106% A.mC.mU. G. G. A. A. G.fU.mC. G. A. G. A.mC.Chl G*mU* A* A* G*mC* C. 14067 993 3245 mC.mC.mU. 3246 P.mC. A. A. A.fC.fU.fU. 67% A.mU.mC. A. A. G. A.fU. A. G. G.mU.mU.mU. G*mC*mU*mU* G* G.Chl G* A. 14068 825 3247 mU. 3248 P.mA. G. 93% G.mU.mU.mC.mC. G.fU.fC.fU.fU. G. G. A. A. A. G. A.mC. A* G* G*mC* A.mC.mC.mU.Chl G*mC* U. 14069 926 3249 mC. G. A. A. 3250 P.mC. A. G. G.fU.fC. A. 95% G.mC.mU. G. G.fC.fU.mU.mC. A.mC.mC.mU. G*mC* A* A* G* G* G.Chl C. 14070 923 3251 mU.mU. G.mC. G. 3252 P.mG.fU.fC. A. 95% A. A. G.mC.mU. G. G.fC.fU.fU.fC. G.mC. A. A.mC.Chl A* G* G*mC*mC*mU* G. 14071 866 3253 mC. A. A.mU. G. 3254 P.mG. G.fC. G.fU.fU. 132% A.mC. A. A.mC. G.fU.fC. A.mU.mU. G* G.mC.mC.Chl G*mU* A* A*mC* C. 14072 563 3255 G.mU. A.mC.mC. 3256 P.mC. G.fU. G.fC. n/a A. G.mU. G.mC. A.fC.fU. G. G.mU. A.mC. G.Chl A.mC*mU*mU* G*mC* A* G. 14073 823 3257 mC.mC.mU. 3258 P.mG.fU.fC.fU.fU. G. 98% G.mU.mU.mC.mC. G. A. A.fC. A. G. A. A. G. A.mC.Chl G*mC* G*mC*mU*mC* C. 14074 1233 3259 mU. A.mC. G. G. 3260 P.mC.fC. A.fU. 109% A. G. A.mC. A.mU. G.fU.fC.fU.fC.fC. G. G.Chl G.mU. A*mC* A*mU*mC*mU* U. 14075 924 3261 mU. G.mC. G. A. 3262 P.mG. G.fU.fC. A. 95% A. G.mC.mU. G. G.fC.fU.fU.fC. G.mC. A.mC.mC.Chl A* A* G* G*mC*mC* U. 14076 921 3263 mC.mC.mU.mU. 3264 P.mC. A. G.fC.fU.fU.fC. 116% G.mC. G. A. A. G.fC. A. A. G. G.mC.mU. G.Chl G*mC*mC*mU* G* A* C. 14077 443 3265 mC.mU. G.mU. G. 3266 P.mG.fC.fC. G. A. A. 110% A.mC.mU.mU.mC. G.fU.fC. A.mC. A. G* G. G.mC.Chl A* A* G* A* G* G. 14078 1041 3267 G.mC.mU. A. A. 3268 P.mC. A.fC. A. G. A. 99% A.mU.mU.mC.mU. A.fU.fU.fU. A. G.mU. G.Chl G.mC*mU*mC* G* G*mU* A. 14079 1042 3269 mC.mU. A. A. 3270 P.mC.fC. A.fC. A. G. A. 109% A.mU.mU.mC.mU. A.fU.fU.mU. A. G.mU. G. G.Chl G*mC*mU*mC* G* G* U. 14080 755 3271 A. G. A.mC. A.mC. 3272 P.mG.fC.fC. A. A. A.fC. 121% G.mU.mU.mU. G. G.fU. G.mC.Chl G.mU.mC.mU*mU*mC* mC* A* G* U. 14081 467 3273 mC.mC. G.mC. A. 3274 P.mG.fC. C.fG. A. 132% A. G. A.mU.mC. G. U.fC.fU.fU.fG. C.mG. G.mC.Chl G*mU*mU* G* G*mC* C. 14082 995 3275 mU. A.mU.mC. A. 3276 P.mC.fU.fC. A. A. 105% A. G.mU.mU.mU. A.fC.fU.fU. G. A.mU. G. A. G.Chl A* G* G*mC*mU*mU* G. 14083 927 3277 G. A. A. 3278 P.mC.fC. A. G. G.fU.fC. 114% G.mC.mU. G. A. G.fC.mU.mU.mC* A.mC.mC.mU. G. G*mC* A* A* G* G. G.Chl 17356 1267 3279 A.mC. A.mU.mU. 3280 P.mU. A.fU. G. A. 120% A. A.mC.mU.mC. G.mU.fU. A. A.fU. A.mU. A.Chl G.fU*fC*fU*fC*fU*fC* A. 17357 1267 3281 G. A.mC. 3282 P.mU. A.fU. G. A. 56% A.mU.mU. A. G.mU.fU. A. A.fU. A.mC.mU.mC. G.fU*fC*fU*fC*fU*fC* A.mU. A.Chl A. 17358 1442 3283 mU. G. A. A. G. A. 3284 P.mU.fU. A. A.fC. 34% A.mU. G.mU.mU. A.fU.fU.fC.fU.fU.fC. A* A. A.Chl A* A*fC*fC* A* G. 17359 1442 3285 mU.mU. G. A. A. 3286 P.mU.fU. A. A.fC. 31% G. A. A.mU. A.fU.fU.fC.fU.fU.fC. A* G.mU.mU. A. A* A*fC*fC* A* G. A.Chl 17360 1557 3287 G. A.mU. A. 3288 P.mU.fU. A. A. G. A.fU. 59% G.mC. G.fC.fU. A.fU.fC*fU* A.mU.mC.mU.mU. G* A*fU* G* A. A. A.Chl 17361 1557 3289 A. G. A.mU. A. 3290 P.mU.fU. A. A. G. A.fU. 47% G.mC. G.fC.fU. A.fU.fC*fU* A.mU.mC.mU.mU. G* A*fU* G* A. A. A.Chl 17362 1591 3291 mU. G. A. A. 3292 P.mU. A. A.fU.fU. A.fC. 120% G.mU. G.mU. A. A.fC.fU.fU.fC. A* A* A.mU.mU. A.Chl A*fU* A* G* C. 17363 1599 3293 A. A.mU.mU. G. 3294 P.mU.fU.fC.fC.fU.fU.fC. 71% A. G. A. A. G. G. A. fU.fC. A. A.fU.fU* A.Chl A*fC* A*fC*fU* U. 17364 1601 3295 mU.mU. G. A. G. 3296 P.mU.fU.fU.fU.fC.fC.fU. 62% A. A. G. G. A. A. A. fU.fC.fU.fC. A. A.Chl A*fU*fU* A*fC* A* C. 17365 1732 3297 mC. 3298 P.mU.fC. G. A. A.fU.fC. 99% A.mU.mU.mC.mU. A. G. A. A.fU. G. A.mU.mU.mC. G*fU*fC* A* G* A* G. G. A.Chl 17366 1734 3299 mU.mU.mC.mU. 3300 P.mU.fU.fU.fC. G. A. 97% G. A.mU.mU.mC. A.fU.fC. A. G. A. A*fU* G. A. A. A.Chl G*fU*fC* A* G. 17367 1770 3301 mC.mU. 3302 P.mU.fU.fC.fU. A. 45% G.mU.mC. G. A.fU.fC. G. A.fC. A. G* A.mU.mU. A. G. A. G* A*fU*fU*fC* C. A.Chl 17368 1805 3303 mU.mU.mU. 3304 P.mU. G.fU.fU. A.fC. A. 71% G.mC.mC.mU. G. G.fC. A. A. G.mU. A. A.mC. A*fU*fU*fC* A*fC* U. A.Chl 17369 1805 3305 A.mU.mU.mU. 3306 P.mU. G.fU.fU. A.fC. A. 67% G.mC.mC.mU. G. G.fC. A. A. G.mU. A. A.mC. A*fU*fU*fC* A*fC* U. A.Chl 17370 1815 3307 A.mC. A. A. 3308 P.mU. A. A.fU.fC.fU. G. 65% G.mC.mC. A. G. G.fC.fU.fU. G.fU*fU* A.mU.mU. A.Chl A*fC* A* G* G. 17371 1815 3309 A. A.mC. A. A. 3310 P.mU. A. A.fU.fC.fU. G. 35% G.mC.mC. A. G. G.fC.fU.fU. G.fU*fU* A.mU.mU. A.Chl A*fC* A* G* G. 17372 2256 3311 mC. A. 3312 P.mU. A.fC. A. A. A.fU. 113% G.mU.mU.mU. A. A. A.fC.fU. A.mU.mU.mU. G*fU*fC*fC* G* A* A. G.mU. A.Chl 17373 2265 3313 mU. G.mU.mU. G. 3314 P.mU. A.fC. 35% A. G. A. G.mU. A.fC.fU.fC.fU.fC. A. G.mU. A.Chl A.fC. A* A* A*fU* A* A* A. 17374 2265 3315 mU.mU. 3316 P.mU. A.fC. 31% G.mU.mU. G. A. A.fC.fU.fC.fU.fC. A. G. A. G.mU. A.fC. A* A* A*fU* A* G.mU. A.Chl A* A. 17375 2295 3317 mU. G.mC. 3318 P.mU.fU. A. G. A. A. A. 34% A.mC.mC.mU.mU. G. G.fU. G.fC. A* A* mU.mC.mU. A. A*fC* A*fU* G. A.Chl 17376 2295 3319 mU.mU. G.mC. 3320 P.mU.fU. A. G. A. A. A. 28% A.mC.mC.mU.mU. G. G.fU. G.fC. A* A* mU.mC.mU. A. A*fC* A*fU* G. A.Chl 17377 1003 3321 mU.mU. G. A. 3322 P.mU.fC. A. G. A. A. A. 67% G.mC.mU.mU.mU. G.fC.fU.fC. A. A* mC.mU. G. A.Chl A*fC*fU*fU* G* A. 17378 2268 3323 mU. G. A. G. A. 3324 P.mU. G.fU.fC. A.fC. 42% G.mU. G.mU. G. A.fC.fU.fC.fU.fC. A* A.mC. A.Chl A*fC* A* A* A* U. 17379 2272 3325 A. G.mU. G.mU. 3326 P.mU.fU.fU.fU. G. 35% G. A.mC.mC. A. A. G.fU.fC. A.fC. A. A.Chl A.fC.fU*fC*fU*fC* A* A* C. 17380 2272 3327 G. A. G.mU. 3328 P.mU.fU.fU.fU. G. 29% G.mU. G. G.fU.fC. A.fC. A.mC.mC. A. A. A. A.fC.fU*fC*fU*fC* A* A.Chl A* C. 17381 2273 3329 G.mU. G.mU. G. 3330 P.mU.fU.fU.fU.fU. G. 42% A.mC.mC. A. A. A. G.fU.fC. A.fC. A. A.Chl A.fC*fU*fC*fU*fC* A* A. 17382 2274 3331 mU. G.mU. G. 3332 P.mU.fC.fU.fU.fU.fU. 42% A.mC.mC. A. A. A. G. G.fU.fC. A.fC. A. G. A.Chl A*fC*fU*fC*fU*fC* A. 17383 2274 3333 G.mU. G.mU. G. 3334 P.mU.fC.fU.fU.fU.fU. 37% A.mC.mC. A. A. A. G. G.fU.fC. A.fC. A. G. A.Chl A*fC*fU*fC*fU*fC* A. 17384 2275 3335 G.mU. G. 3336 P.mU. 24% A.mC.mC. A. A. A. A.fC.fU.fU.fU.fU. G. A. G.mU. A.Chl G.fU.fC. A.fC* A*fC*fU*fC*fU* C. 17385 2277 3337 G. A.mC.mC. A. A. 3338 P.mU.fU. A. 27% A. A. G.mU.mU. A. A.fC.fU.fU.fU.fU. G. A.Chl G.fU.fC* A*fC* A*fC*fU* C. 17386 2296 3339 G.mC. 3340 P.mU.fC.fU. A. G. A. A. 23% A.mC.mC.mU.mU. A. G. G.fU. G.fC* A* mU.mC.mU. A. G. A* A*fC* A* U. A.Chl 17387 2299 3341 mC.mC.mU.mU.mU. 3342 P.mU.fC. A. A.fC.fU. A. 46% mC.mU. A. G. A. A. A. G. G*fU* G.mU.mU. G. G*fC* A* A* A. A.Chl 21138 2296 3343 G.mC. 3344 P.mU.fC.fU. A. G. 42% A.mC.mC.mU.mU. A.mA. A. G. G.fU. mU.mC.mU. A. G. G.mC* A* A* A*mC* A.TEG-Chl A* U. 21139 2296 3345 G.mC. 3346 P.mU.fC.fU. A. G.mA. 32% A.mC.mC.mU.mU. A.mA. G. G.fU. G.mC* mU.mC.mU. A. G. A* A* A*mC* A* U. A.TEG-Chl 21140 2296 3347 G.mC. 3348 P.mU.fC.fU. A. G. A. A. 41% A.mC.mC.mU.mU. A. G. G.fU. G.mC* mU.mC.mU. A. G. A*mA* A*mC* A* U. A.TEG-Chl 21141 2296 3349 G.mC. 3350 P.mU.fC.fU. A. G. 51% A.mC.mC.mU.mU. A.mA. A. G. G.fU. mU.mC.mU. A. G. G.mC* A*mA* A*mC* A.TEG-Chl A* U. 21142 2296 3351 G.mC. 3352 P.mU.fC.fU. A. G.mA. 25% A.mC.mC.mU.mU. A.mA. G. G.fU. G.mC* mU.mC.mU. A. G. A*mA* A*mC* A* U. A.TEG-Chl 21143 2296 3353 G.mC. 3354 P.mU.fC.fU. A. G. A. A. 61% A.mC.mC.mU.mU. A. G. G.fU. mU.mC.mU. A. G. G.fC*mA*mA*mA*fC* A.TEG-Chl mA* U. 21144 2296 3355 G.mC. 3356 P.mU.fC.fU. A. G. 49% A.mC.mC.mU.mU. A.mA. A. G. G.fU. mU.mC.mU. A. G. G.fC*mA*mA*mA*fC* A.TEG-Chl mA* U. 21145 2296 3357 G.mC. 3358 P.mU.fC.fU. A. G.mA. 46% A.mC.mC.mU.mU. A.mA. G. G.fU. mU.mC.mU. A. G. G.fC*mA*mA*mA*fC* A.TEG-Chl mA* U. 21146 2296 3359 G.mC. 3360 P.mU.fC.fU. A. G. A. A. 37% A.mC.mC.mU.mU. A. G. G.fU. G.fC* A* mU.mC.mU. A* A*fC* A* U. A*mG*mA.TEG- Chl 21147 2296 3361 mG*mC* 3362 P.mU.fC.fU. A. G. A. A. 43% A.mC.mC.mU.mU. A. G. G.fU. G.fC* A* mU.mC.mU. A* A*fC* A* U. A*mG*mA.TEG- Chl 21148 2296 3363 mG*mC*mA.mC. 3364 P.mU.fC.fU. A. G. A. A. 29% mC.mU.mU.mU.mC. A. G. G.fU. G.fC* A* mU.mA*mG*mA. A* A*fC* A* U. TEG-Chl 21149 2275 3365 G.mU. G. 3366 P.mU. 138% A.mC.mC. A. A. A. A.fC.fU.fU.fU.fU. G. A. G.fU.fC. A.fC* G*mU*mA.TEG- A*fC*fU*fC*fU* C. Chl 21150 2275 3367 mG*mU* G. 3368 P.mU. 116% A.mC.mC. A. A.fC.fU.fU.fU.fU. G. A.mA. A. G.fU.fC. A.fC* G*mU*mA.TEG- A*fC*fU*fC*fU* C. Chl 21151 2275 3369 mG*mU*mG.mA. 3370 P.mU. 105% mC.mC.mA.mA.mA. A.fC.fU.fU.fU.fU. G. mA.mG*mU*mA. G.fU.fC. A.fC* TEG-Chl A*fC*fU*fC*fU* C. 21152 2295 3371 mU.mU. G.mC. 3372 P.mU.fU. A. G. A.mA. 46% A.mC.mC.mU.mU. A. G. G.fU. G.fC. A. A* mU.mC.mU. A. A*fC* A*fA* G* G. A.TEG-Chl 21153 2295 3373 mU.mU. G.mC. 3374 P.mU.fU. A. G.mA. 28% A.mC.mC.mU.mU. A.mA. G. G.fU. G.fC. A. mU.mC.mU. A. A* A*fC* A*fA* G* G. A.TEG-Chl 21154 2295 3375 mU.mU. G.mC. 3376 P.mU.fU.mA. G.mA. 28% A.mC.mC.mU.mU. A.mA. G.mG.fU. G.fC. mU.mC.mU. A. A. A* A*fC* A*fA* G* A.TEG-Chl G. 21155 2295 3377 mU.mU. G.mC. 3378 P.mU.fU. A. G. A.mA. 60% A.mC.mC.mU.mU. A. G. G.fU. G.mC. A. mU.mC.mU. A. A* A*mC* A*mA* G* A.TEG-Chl G. 21156 2295 3379 mU.mU. G.mC. 3380 P.mU.fU. A. G. A.mA. 54% A.mC.mC.mU.mU. A. G. G.fU. G.fC. mU.mC.mU. A. A.mA*mA*fC*mA*fA* A.TEG-Chl mG* G. 21157 2295 3381 mU.mU. G.mC. 3382 P.mU.fU. A. G. A.mA. 40% A.mC.mC.mU.mU. A. G. G.fU. mU.mC.mU. A. G.fC.mA.mA*mA*fC* A.TEG-Chl mA*fA*mG* G. 21158 2295 3383 mU.mU. G.mC. 3384 P.mU.fU. A. G. A.mA. n/a A.mC.mC.mU.mU. A. G. G.fU. G.fC. mU.mC.mU. A. A.mA*mA*fC*mA*mA* A.TEG-Chl mG* G. 21159 2295 3385 mU.mU. G.mC. 3386 P.mU.fU. A. G. A.mA. 41% A.mC.mC.mU.mU. A. G. G.fU. G.fC. mU.mC.mU. A. A.mA*mA*mC*mA*mA* A.TEG-Chl mG* G. 21160 2295 3387 mU.mU. G.mC. 3388 P.mU.fU. A. G. A.mA. 65% A.mC.mC.mU.mU. A. G. G.fU. G.fC.mA. mU.mC.mU. A. A*mA*mC*mA*mA* A.Chl-TEG mG*mG. 21161 2295 3389 mU.mU. G.mC. 3390 P.mU.fU. A. G. A.mA. 43% A.mC.mC.mU.mU. A. G. G.fU. G.fC. A. A* mU.mC.mU. A. A*fC* A*mA*mG* G. A.TEG-Chl 21162 2295 3391 mU.mU. G.mC. 3392 P.mU.fU. A. G. A.mA. 41% A.mC.mC.mU.mU. A. G. G.fU. G.fC.mA. mU.mC.mU. A. A*mA*fC* A.TEG-Chl A*mA*mG* G. 21163 2295 3393 mU.mU. G.mC. 3394 P.mU.fU. A. G. A. A. A. 32% A.mC.mC.mU.mU. G. G.fU. G.fC. A. A* mU.mC.mU. A* A*fC* A* A* G* G. A*TEG-Chl 21164 2295 3395 mU.mU. G.mC. 3396 P.mU.fU. A. G. A. A. A. 39% A.mC.mC.mU.mU. G. G.fU. G.fC. A. A* mU.mC.mU.mA* A*fC* A* A* G* G. mA*TEG-Chl 21165 2295 3397 mU*mU* G.mC. 3398 P.mU.fU. A. G. A. A. A. 28% A.mC.mC.mU.mU. G. G.fU. G.fC. A. A* mU.mC.mU.mA* A*fC* A* A* G* G. mA*TEG-Chl 21166 2295 3399 mU.mU.mG.mC.mA. 3400 P.mU.fU. A. G. A. A. A. 27% mC.mC.mU.mU. G. G.fU. G.fC. A. A* mU.mC.mU.mA* A*fC* A* A* G* G. mA*TEG-Chl 21167 2299 3401 mC.mC.mU.mU.mU. 3402 P.mU.fC. A. A.fC.fU. A. 49% mC.mU. A. G. A.mA. A. G. G*fU* G.mU.mU. G. G*fC* A* A* A. A.TEG-Chl 21168 2299 3403 mC.mC.mU.mU.mU. 3404 P.mU.fC. A. A.fC.fU. A. 53% mC.mU. A. G. A.mA. A. G. G*mU* G.mU.mU. G. G*mC* A* A* A. A.TEG-Chl 21169 2299 3405 mC.mC.mU.mU.mU. 3406 P.mU.fC. A. A.fC.fU. A. 47% mC.mU. A. G.mA. A. A.mG. G*fU* G.mU.mU. G. G*fC* A* A* A. A.TEG-Chl 21170 2299 3407 mC.mC.mU.mU.mU. 3408 P.mU.fC. A. A.fC.fU. A. 70% mC.mU. A. G.mA. A. A.mG. G.mU.mU. G. G*mU* G*mC* A* A* A.TEG-Chl A. 21171 2299 3409 mC.mC.mU.mU.mU. 3410 P.mU.fC. A. A.fC.fU. A. 65% mC.mU. A. G. A.mA. A. G. G*mU* G.mU.mU. G. G*mC* A*mA* A. A.TEG-Chl 21172 2299 3411 mC.mC.mU.mU.mU. 3412 P.mU.fC. A. A.fC.fU. A. 43% mC.mU. A. G. A.mA. A. G. G*mU* G.mU.mU. G. G*mC*mA*mA* A. A.TEG-Chl 21173 2299 3413 mC.mC.mU.mU.mU. 3414 P.mU.fC. A. A.fC.fU. A. 52% mC.mU. A. G. A.mA. A. G.mU.mU. G. G.mG*mU*mG*mC* A.TEG-Chl mA*mA* A. 21174 2299 3415 mC.mC.mU.mU.mU. 3416 P.mU.fC. A. A.fC.fU. A. 47% mC.mU. A. G. A.mA. A. G. G.mU.mU. G. G*mU*mG*mC*mA* A.TEG-Chl mA* A. 21175 2299 3417 mC.mC.mU.mU.mU. 3418 P.mU.fC. A. A.fC.fU. A. 35% mC.mU. A. G. A.mA. A. G. G.mU.mU. G. G*fU*mG*fC*mA*mA* A.TEG-Chl A. 21176 2299 3419 mC.mC.mU.mU.mU. 3420 P.mU.fC. A. A.fC.fU. A. 50% mC.mU. A. G.mA. A. A.mG. G.mU.mU. G. G*fU*mG*fC*mA*mA* A.TEG-Chl A. 21177 2299 3421 mC.mC.mU.mU.mU. 3422 P.mU.fC. A. A.fC.fU. A. 37% mC.mU. A. G. A. A. A. G. G*fU* G.mU.mU*mG*mA. G*fC* A* A* A. TEG-Chl 21178 2299 3423 mC*mC*mU.mU. 3424 P.mU.fC. A. A.fC.fU. A. 36% mU.mC.mU. A. G. A. A. A. G. G*fU* G.mU.mU*mG*mA. G*fC* A* A* A. TEG-Chl 21179 2299 3425 mC*mC*mU.mU. 3426 P.mU.fC. A. A.fC.fU. A. 35% mU.mC.mU.mA.mG. G. A. A. A. G. G*fU* mU.mU*mG*mA. G*fC* A* A* A. TEG-Chl 21203 2296 3427 G.mC. 3428 P.mU.fC.fU. A. G. 40% A.mC.mC.mU.mU. A.mA. A. G. G.fU. mU.mC.mU. G.mC* A* A* A*mC* A*mG*mA.TEG- A* U. Chl 21204 2296 3429 G.mC. 3430 P.mU.fC.fU. A. G.mA. 28% A.mC.mC.mU.mU. A.mA. G. G.fU. G.mC* mU.mC.mU. A* A* A*mC* A* U. A*mG*mA.TEG- Chl 21205 2296 3431 G.mC. 3432 P.mU.fC.fU. A. G.mA. 51% A.mC.mC.mU.mU. A.mA. G. G.fU. G.mC* mU.mC.mU. A*mA* A*mC* A* U. A*mG*mA.TEG- Chl 21206 2296 3433 mG*mC* 3434 P.mU.fC.fU. A. G. 46% A.mC.mC.mU.mU. A.mA. A. G. G.fU. mU.mC.mU. G.mC* A* A* A*mC* A*mG*mA.TEG- A* U. Chl 21207 2296 3435 mG*mC* 3436 P.mU.fC.fU. A. G.mA. 29% A.mC.mC.mU.mU. A.mA. G. G.fU. G.mC* mU.mC.mU. A* A* A*mC* A* U. A*mG*mA.TEG- Chl 21208 2296 3437 mG*mC* 3438 P.mU.fC.fU. A. G.mA. 72% A.mC.mC.mU.mU. A.mA. G. G.fU. G.mC* mU.mC.mU. A*mA* A*mC* A* U. A*mG*mA.TEG- Chl 21209 2296 3439 mG*mC*mA.mC. 3440 P.mU.fC.fU. A. G. 89% mC.mU.mU.mU.mC. A.mA. A. G. G.fU. mU.mA*mG*mA. G.mC* A* A* A*mC* TEG-Chl A* U. 21210 2296 3441 mG*mC*mA.mC. 3442 P.mU.fC.fU. A. G.mA. 65% mC.mU.mU.mU.mC. A.mA. G. G.fU. G.mC* mU.mA*mG*mA. A* A* A*mC* A* U. TEG-Chl 21211 2296 3443 mG*mC*mA.mC. 3444 P.mU.fC.fU. A. G.mA. 90% mC.mU.mU.mU.mC. A.mA. G. G.fU. G.mC* mU.mA*mG*mA. A*mA* A*mC* A* U. TEG-Chl 21212 2295 3445 mU.mU. G.mC. 3446 P.mU.fU. A. G. A.mA. 60% A.mC.mC.mU.mU. A. G. G.fU. mU.mC.mU*mA* G.fC.mA.mA*mA*fC* mA.TEG-Chl mA*mA*mG* G. 21213 2295 3447 mU.mU. G.mC. 3448 P.mU.fU. A. G. A.mA. 63% A.mC.mC.mU.mU. A. G. G.fU. G.fC. mU.mC.mU*mA* A.mA*mA*mC*mA*mA* mA.TEG-Chl mG* G. 21214 2295 3449 mU.mU. G.mC. 3450 P.mU.fU. A. G. A.mA. 52% A.mC.mC.mU.mU. A. G. G.fU. G.fC. A. A* mU.mC.mU*mA* A*fC* A*mA*mG* G. mA.TEG-Chl 21215 2295 3451 mU.mU. G.mC. 3452 P.mU.fU. A. G. A.mA. 45% A.mC.mC.mU.mU. A. G. G.fU. G.fC.mA. mU.mC.mU*mA* A*mA*fC* mA.TEG-Chl A*mA*mG* G. 21216 2295 3453 mU*mU* G.mC. 3454 P.mU.fU. A. G. A.mA. 65% A.mC.mC.mU.mU. A. G. G.fU. mU.mC.mU*mA* G.fC.mA.mA*mA*fC* mA.TEG-Chl mA*mA*mG* G. 21217 2295 3455 mU*mU* G.mC. 3456 P.mU.fU. A. G. A.mA. 69% A.mC.mC.mU.mU. A. G. G.fU. G.fC. mU.mC.mU*mA* A.mA*mA*mC*mA*mA* mA.TEG-Chl mG* G. 21218 2295 3457 mU*mU* G.mC. 3458 P.mU.fU. A. G. A.mA. 62% A.mC.mC.mU.mU. A. G. G.fU. G.fC. A. A* mU.mC.mU*mA* A*fC* A*mA*mG* G. mA.TEG-Chl 21219 2295 3459 mU*mU* G.mC. 3460 P.mU.fU. A. G. A.mA. 54% A.mC.mC.mU.mU. A. G. G.fU. G.fC.mA. mU.mC.mU*mA* A*mA*fC* mA.TEG-Chl A*mA*mG* G. 21220 2295 3461 mU.mU.mG.mC.mA. 3462 P.mU.fU. A. G. A.mA. 52% mC.mC.mU.mU. A. G. G.fU. mU.mC.mU*mA* G.fC.mA.mA*mA*fC* mA.TEG-Chl mA*mA*mG* G. 21221 2295 3463 mU.mU.mG.mC.mA. 3464 P.mU.fU. A. G. A.mA. 53% mC.mC.mU.mU. A. G. G.fU. G.fC. mU.mC.mU*mA* A.mA*mA*mC*mA*mA* mA.TEG-Chl mG* G. 21222 2295 3465 mU.mU.mG.mC.mA. 3466 P.mU.fU. A. G. A.mA. 43% mC.mC.mU.mU. A. G. G.fU. G.fC. A. A* mU.mC.mU*mA* A*fC* A*mA*mG* G. mA.TEG-Chl 21223 2295 3467 mU.mU.mG.mC.mA. 3468 P.mU.fU. A. G. A.mA. 43% mC.mC.mU.mU. A. G. G.fU. G.fC.mA. mU.mC.mU*mA* A*mA*fC* mA.TEG-Chl A*mA*mG* G. 21224 2299 3469 mC.mC.mU.mU.mU. 3470 P.mU.fC. A. A.fC.fU. A. 60% mC.mU. A. G. A.mA. A. G. G.mU.mU*mG*mA. G*fU*mG*fC*mA*mA* TEG-Chl A. 21225 2299 3471 mC*mC*mU.mU. 3472 P.mU.fC. A. A.fC.fU. A. 67% mU.mC.mU. A. G. A.mA. A. G. G.mU.mU*mG*mA. G*fU*mG*fC*mA*mA* TEG-Chl A. 21226 2299 3473 mC*mC*mU.mU. 3474 P.mU.fC. A. A.fC.fU. A. 66% mU.mC.mU.mA.mG. G. A.mA. A. G. mU.mU*mG*mA. G*fU*mG*fC*mA*mA* TEG-Chl A. 21227 2296 3475 G.mC. 3476 P.mU.fC.fU. A. G.mA. 49% A.mC.mC.mU.mU. A.mA. G. G.fU. mU.mC.mU. G.fC*mA*mA*mA*fC* A*mG*mA.TEG- mA* U. Chl 20584 2296 3477 G.mC. 3478 P.mU.fC.fU. A. G. A. A. 70% A.mC.mC.mU.mU. A. G. G.mU. G.mC* A* mU.mC.mU. A. G. A* A*mC* A* U. A.Chl-TEG 20585 2296 3479 G.mC. 3480 P.mU.fC.fU. A. G. A. A. 15% A.mC.mC.mU.mU. A. G. G.fU. G.mC* A* mU.mC.mU. A. G. A* A*mC* A* U. A.Chl-TEG 20586 2296 3481 G.mC. 3482 P.mU. C. U. A. G. A. A. 30% A.mC.mC.mU.mU. A. G. G.mU. G.mC* A* mU.mC.mU. A. G. A* A*mC* A* U. A.Chl-TEG 20587 2296 3483 G.mC. 3484 P.mU.fC.fU. A. G. A. A. 32% A.mC.mC.mU.mU. A. G. G.fU. mU.mC.mU. A. G. G.fC*mA*mA*mA*fC* A.Chl-TEG mA* U. 20616 2275 3485 G.mU. G. 3486 P.mU. 22% A.mC.mC. A. A. A. A.fC.fU.fU.fU.fU. G. A. G.mU. A.Chl- G.fU.mC. A.mC* TEG A*mC*mU*mC*mU* C. 20617 2275 3487 G.mU. G. 3488 P.mU. 18% A.mC.mC. A. A. A. A.fC.fU.fU.fU.fU. G. A. G.mU. A.Chl- G.fU.fC. A.mC* TEG A*fC*mU*fC*mU* C. 20618 2275 3489 G.mU. G. 3490 P.mU. A. C. U. U. U. U. 36% A.mC.mC. A. A. A. G. G. U.mC. A.mC* A. G.mU. A.Chl- A*mC*mU*mC*mU* TEG C. 20619 2275 3491 G.mU. G. 3492 P.mU. 28% A.mC.mC. A. A. A. A.fC.fU.fU.fU.fU. G. A. G.mU. A.Chl- G.fU.fC. TEG A.mC*mA*mC*mU*mC* mU* C. 21381 2275 3493 G.mU. G. 3494 P.mU. 28% A.mC.mC. A. A. A. A.fC.fU.fU.fU.fU. G. A. G.fU.mC. A.mC* G*mU*mA.TEG- A*mC*mU*mC*mU* Chl C. 21382 2275 3495 G.mU. G. 3496 P.mU. 28% A.mC.mC. A. A. A. A.fC.fU.fU.fU.fU. G. A. G.fU.fC. A.mC* G*mU*mA.TEG- A*fC*mU*fC*mU* C. Chl 21383 2275 3497 mG*mU*mG.mA. 3498 P.mU. 43% mC.mC.mA.mA.mA. A.fC.fU.fU.fU.fU. G. mA.mG*mU*mA. G.fU.mC. A.mC* TEG-Chl A*mC*mU*mC*mU* C. 21384 2275 3499 mG*mU*mG.mA. 3500 P.mU. 50% mC.mC.mA.mA.mA. A.fC.fU.fU.fU.fU. G. mA.mG*mU*mA. G.fU.fC. A.mC* TEG-Chl A*fC*mU*fC*mU* C. 20392 2275 3501 G.mU. G. 3502 P.mU. 28% A.mC.mC. A. A. A. A.fC.fU.fU.fU.fU. G. A. G.mU. A.TEG- G.fU.fC. A.fC* Chl A*fC*fU*fC*fU* C. 20393 2296 3503 G.mC. 3504 P.mU.fC.fU. A. G. A. A. 35% A.mC.mC.mU.mU. A. G. G.fU. G.fC* A* mU.mC.mU. A. G. A* A*fC* A* U. A.TEG-Chl 21429 2275 3505 G.mU. G. 3506 P.mU. 36% A.mC.mC. A. A. A. A.fC.fU.fU.fU.fU. G. A. G.fU.fC. A.mC* G*mU*mA.Teg- A*fC*mU*fC*mU* C. Chl 21430 2275 3507 G.mU. G. 3508 P.mU. 31% A.mC.mC. A. A.fC.fU.fU.fU.fU. G. A.mA. A. G.fU.mC. A.mC* G*mU*mA.Teg- A*mC*mU*mC*mU* Chl C.

TABLE 21 Inhibition of gene expression with TGFB2 sd-rxRNA sequences (Accession Number: NM_001135599.1) % remaining Oligo Start SEQ SEQ ID expression (1 Number Site ID NO Sense sequence NO Antisense sequence uM, A549) 14408 1324 3509 G. 3510 P.mU.fC. G. A. A. G. G. 94% G.mC.mU.mC.mU. A. G. A. G.mC.mC* mC.mC.mU.mU.mC. A*mU*mU*mC* G* C. G. A.Chl 14409 1374 3511 G. A.mC. A. G. G. A. 3512 P.mC.fC. A. G. n/a A.mC.mC.mU. G. G.fU.fU.fC.fC.fU. G.Chl G.mU.mC*mU*mU*mU* A*mU* G. 14410 946 3513 mC.mC. A. A. G. G. 3514 P.mU. A. A. 90% A. G. A.fC.fC.fU.fC.fC.fU.mU. G.mU.mU.mU. G. G*mC* G*mU* A* A.Chl G* U. 14411 849 3515 A.mU.mU.mU.mC. 3516 P.mU. G.fU. A. G. A.fU. 72% mC. A.mU.mC.mU. G. G. A. A. A.mU*mC* A.mC. A.Chl A*mC*mC*mU* C. 14412 852 3517 mU.mC.mC. 3518 P.mU. G.fU.fU. G.fU. 76% A.mU.mC.mU. A. G. A.fU. G. G. A* A* A.mC. A. A.mC. A*mU*mC* A* C. A.Chl 14413 850 3519 mU.mU.mU.mC.mC. 3520 P.mU.fU. G.fU. A. G. 98% A.mU.mC.mU. A.fU. G. G. A. A. A.mC. A. A.Chl A*mU*mC* A*mC*mC* U. 14414 944 3521 mC. G.mC.mC. A. A. 3522 P.mA. 100%  G. G. A. G. A.fC.fC.fU.fC.fC.fU.fU. G.mU.mU.Chl G. G.mC. G*mU* A* G*mU* A* C. 14415 1513 3523 G.mU. G. G.mU. G. 3524 P.mU.fU.fC.fU. G. n/a A.mU.mC. A. G. A. A.fU.fC. A.fC.mC. A.Chl A.mC*mU* G* G*mU* A* U. 14416 1572 3525 mC.mU.mC.mC.mU. 3526 P.mA.fC. A.fU.fU. A. 100%  G.mC.mU. A. G.fC. A. G. G. A. G* A.mU. G.mU.Chl A*mU* G*mU* G* G. 14417 1497 3527 A.mC.mC.mU.mC.mC. 3528 P.mU. A.fU. A.fU. 73% A.mC. A.mU. G.fU. G. G. A. G. A.mU. A.Chl G.mU* G*mC*mC* A*mU* C. 14418 1533 3529 A. A. G.mU.mC.mC. 3530 P.mU.fC.fC.fU. A. G.fU. 98% A.mC.mU. A. G. G. G. G. A.Chl A.mC.mU.mU*mU* A*mU* A* G* U. 14419 1514 3531 mU. G. G.mU. G. 3532 P.mU.fU.fU.fC.fU. G. 86% A.mU.mC. A. G. A. A.fU.fC. A.mC.mC. A. A.Chl A*mC*mU* G* G*mU* A. 14420 1534 3533 A. G.mU.mC.mC. 3534 P.mU.fU.fC.fC.fU. A. 99% A.mC.mU. A. G. G. G.fU. G. G. A. A.Chl A.mC.mU*mU*mU* A*mU* A* G. 14421 943 3535 A.mC. G.mC.mC. A. 3536 P.mA.fC.fC.fU.fC.fC.fU. 41% A. G. G. A. G. fU. G. G.mC. G.mU* G.mU.Chl A* G*mU* A*mC* U. 18570 2445 3537 mU. A.mU.mU.mU. 3538 P.mU. A.fC. A.fC. A. 79% A.mU.mU. G.mU. A.fU. A. A. A.fU. A* G.mU. A.Chl A*fC*fU*fC* A* C. 18571 2445 3539 mU.mU. 3540 P.mU. A.fC. A.fC. A. 75% A.mU.mU.mU. A.fU. A. A. A.fU. A* A.mU.mU. G.mU. A*fC*fU*fC* A* C. G.mU. A.Chl 18572 2083 3541 A.mU. C. A. G.mU. 3542 P.mU.fU.fU.fU. A. 47% G.mU.mU. A. A. A. A.fC. A.fC.fU. G. A.fU* A.Chl G* A* A*fC*fC* A. 18573 2083 3543 mC. A.mU.mC. A. 3544 P.mU.fU.fU.fU. A. 17% G.mU. G.mU.mU. A.fC. A.fC.fU. G. A.fU* A. A. A. A.Chl G* A* A*fC*fC* A. 18574 2544 3545 A.mU. G. 3546 P.mU.fU.fC.fC.fU.fU. 59% G.mC.mU.mU. A. A. A. A. G.fC.fC. A. G. G. A. A.Chl U*fC*fC* A*fU* G* A. 18575 2544 3547 G. A.mU. G. 3548 P.mU.fU.fC.fC.fU.fU. 141%  G.mC.mU.mU. A. A. A. A. G.fC.fC. A. G. G. A. A.Chl U*fC*fC* A*fU* G* A. 18576 2137 3549 mU.mU. G.mU. 3550 P.mU. A. A.fC. A. G. A. 77% G.mU.mU.mC.mU. A.fC. A.fC. A. A* G.mU.mU. A.Chl A*fC*fU*fU*fC* C. 18577 2137 3551 mU.mU.mU. G.mU. 3552 P.mU. A. A.fC. A. G. A. 59% G.mU.mU.mC.mU. A.fC. A.fC. A. A* G.mU.mU. A.Chl A*fC*fU*fU*fC* C. 18578 2520 3553 A. A. A.mU. 3554 P.mU. G. G.fC. A. A. A. 75% A.mC.mU.mU.mU. G.fU. A.fU.fU.fU* G* G.mC.mC. A.Chl G*fU*fC*fU* C. 18579 2520 3555 mC. A. A. A.mU. 3556 P.mU. G. G.fC. A. A. A. 55% A.mC.mU.mU.mU. G.fU. A.fU.fU.fU* G* G.mC.mC. A.Chl G*fU*fC*fU* C. 18580 3183 3557 mC.mU.mU. G.mC. 3558 P.mU.fU.fU. G.fU. A. 84% A.mC.mU. A.mC. A. G.fU. G.fC. A. A. A. A.Chl G*fU*fC* A* A* A* C. 18581 3183 3559 A.mC.mU.mU. 3560 P.mU.fU.fU. G.fU. A. 80% G.mC. A.mC.mU. G.fU. G.fC. A. A. A.mC. A. A. A.Chl G*fU*fC* A* A* A* C. 18582 2267 3561 G. A. 3562 P.mU. A.fC.fU. A. A.fU. 82% A.mU.mU.mU. A. A. A.mU.mU. A. G.mU. A.fU.fU.fC*fU*fU*fC*fC* A.Chl A* G. 18583 2267 3563 A. G. A. 3564 P.mU. A.fC.fU. A. A.fU. 67% A.mU.mU.mU. A. A. A.mU.mU. A. G.mU. A.fU.fU.fC*fU*fU*fC*fC* A.Chl A* G. 18584 3184 3565 mU.mU. G.mC. 3566 P.mU.fU.fU.fU. G.fU. 77% A.mC.mU. A.mC. A. A. G.fU. G.fC. A. A* A. A. A.Chl G*fU*fC* A* A* A. 18585 3184 3567 mC.mU.mU. G.mC. 3568 P.mU.fU.fU.fU. G.fU. 59% A.mC.mU. A.mC. A. A. G.fU. G.fC. A. A* A. A. A.Chl G*fU*fC* A* A* A. 18586 2493 3569 A.mU. A. A. A. 3570 P.mU.fC. A.fC.fC.fU. 84% A.mC. A. G. G.mU. G.fU.fU.fU.fU. G. A.Chl A.fU*fU*fU*fU*fC*fC* A. 18587 2493 3571 A. A.mU. A. A. A. 3572 P.mU.fC. A.fC.fC.fU. 70% A.mC. A. G. G.mU. G.fU.fU.fU.fU. G. A.Chl A.fU*fU*fU*fU*fC*fC* A. 18588 2297 3573 G. A.mC. A. A.mC. 3574 P.mU. G.fU.fU. 40% A. A.mC. A. A.mC. G.fU.fU. G.fU.fU. A.Chl G.fU.fC* G*fU*fU* G*fU* U. 18589 2046 3575 A.mU. G. 3576 P.mU.fU. G.fU.fU. 39% C.mU.mU. G.mU. A. A.fC. A. A. G.fC. A.mC. A. A.Chl A.fU*fC* A*fU*fC* G* U. 18590 2531 3577 mC. A. G. A. A. 3578 P.mU.fC. A.fU. G. A. 56% A.mC.mU.mC. G.fU.fU.fU.fC.fU. G* A.mU. G. A.Chl G*fC* A* A* A* G. 18591 2389 3579 G.mU. A.mU.mU. 3580 P.mU. G.fC. A.fU. A. 64% G.mC.mU. A.mU. G.fC. A. A.fU. A.fC* A* G.mC. A.Chl G* A* A* A* A. 18592 2530 3581 mC.mC. A. G. A. A. 3582 P.mU. A.fU. G. A. 44% A.mC.mU.mC. G.fU.fU.fU.fC.fU. G. A.mU. A.Chl G*fC* A* A* A* G* U. 18593 2562 3583 A.mC.mU.mC. A. A. 3584 P.mU. G.fC.fU.fC. 87% A.mC. G. A. G.mC. G.fU.fU.fU. G. A. A.Chl G.fU*fU*fC* A* A* G* U. 18594 2623 3585 A.mU. A.mU. G. 3586 P.mU.fU.fC.fU.fC. G. 69% A.mC.mC. G. A. G. G.fU.fC. A.fU. A.fU* A. A.Chl A* A*fU* A* A* C. 18595 2032 3587 mC. G. A.mC. G. 3588 P.mU.fU.fC. G.fU.fU. 55% A.mC. A. A.mC. G. G.fU.fC. G.fU.fC. A. A.Chl G*fU*fC* A*fU*fC* A. 18596 2809 3589 G.mU. A. A. 3590 P.mU.fU.fC. A.fC.fU. G. 58% A.mC.mC. A. G.mU. G.fU.fU.fU. A.fC*fU* G. A. A.Chl A* A* A*fC* U. 18597 2798 3591 mU.mU. G.mU.mC. 3592 P.mU.fC.fU. A. A. 38% A. G.mU.mU.mU. A. A.fC.fU. G. A.fC. A. A* G. A.Chl A* G* A* A*fC* C. 18598 2081 3593 mU.mC. A.mU.mC. 3594 P.mU.fU. A. A.fC. 25% A. G.mU. A.fC.fU. G. A.fU. G. A* G.mU.mU. A. A.Chl A*fC*fC* A* A* G. 18599 2561 3595 A. A.mC.mU.mC. A. 3596 P.mU.fC.fU.fC. 57% A. A.mC. G. A. G. G.fU.fU.fU. G. A. A.Chl G.fU.fU*fC* A* A* G*fU* U. 18600 2296 3597 mC. G. A.mC. A. 3598 P.mU.fU.fU. G.fU.fU. 69% A.mC. A. A.mC. A. G.fU.fU. G.fU.fC. A. A.Chl G*fU*fU* G*fU*fU* C. 18601 2034 3599 A.mC. G. A.mC. A. 3600 P.mU.fC. A.fU.fC. 22% A.mC. G. A.mU. G. G.fU.fU. G.fU.fC. A.Chl G.fU*fC* G*fU*fC* A*fU. 18602 2681 3601 G.mC.mU. 3602 P.mU.fU.fC.fC.fU.fU. 43% G.mC.mC.mU. A. A. A. G. G.fC. A. G.fC*fU* G. G. A. A.Chl G* A*fU* A* C. 18603 2190 3603 A.mU.mU.mC.mU. 3604 P.mU. G. A. A. A.fU. 128%  A.mC. G.fU. A. G. A. A.fU* A* A.mU.mU.mU.mC. A* G* G*fC* C. A.Chl 20604 2083 3605 mC. A.mU.mC. A. 3606 P.mU.fU.fU.fU. A. 19% G.mU. G.mU.mU. A.fC. A.fC.fU. G. A. A. A. A.Chl A.mU* G* A* A*mC*mC* A. 20605 2083 3607 mC. A.mU.mC. A. 3608 P.mU.fU.fU.fU. A. 20% G.mU. G.mU.mU. A.fC. A.fC.fU. G. A. A. A. A.Chl A.mU* G* A* A*fC*mC* A. 20606 2083 3609 mC. A.mU.mC. A. 3610 P.mU. U. U. U. A. A. C. 82% G.mU. G.mU.mU. A. C. U. G. A.mU* G* A. A. A. A.Chl A* A*mC*mC* A. 20607 2083 3611 mC. A.mU.mC. A. 3612 P.mU.fU.fU.fU. A. 59% G.mU. G.mU.mU. A.fC. A.fC.fU. G. A. A. A. A.Chl A.fU*mG*mA*mA*fC* fC* A. 21722 2081 3613 mU.mC. A.mU.mC. 3614 P.mU.fU. A. A.fC. 34% A. G.mU. A.fC.fU. G. A.fU. G. A* G.mU.mU. A. A.Chl A*mC*mC* A* A* G. 21723 2081 3615 mU.mC. A.mU.mC. 3616 P.mU.fU. A. A.fC. 53% A. G.mU. A.fC.fU. G. A.fU. G.mU.mU. A. A.Chl G.mA*mA*mC*mC*mA* mA* G. 21724 2081 3617 mU.mC. A.mU.mC. 3618 P.mU.fU. A. A.fC. 48% A. G.mU. A.fC.fU. G. A.mU. G.mU.mU. A. A.Chl G.mA*mA*mC*mC*mA* mA* G. 21725 2081 3619 mU.mC. A.mU.mC. 3620 P.mU.fU. A. A.fC. 45% A. G.mU. A.fC.fU. G. A.fU. G. A* G.mU.mU. A. A.Chl A*fC*fC*mA*mA* G. 21726 2081 3621 mU.mC. A.mU.mC. 3622 P.mU.fU. A. A.fC. 54% A. G.mU. A.fC.fU. G. A.fU. G.mU.mU. A. A.Chl G.mA*mA*fC*fC*mA* mA* G. 21727 2081 3623 mU.mC. A.mU.mC. 3624 P.mU.fU.A. A.fC. 29% A. G.mU. A.fC.fU. G. A.fU. G. A* G.mU.mU*mA*mA. A*fC*fC* A* A* G. TEG-Chl 21728 2081 3625 mU*mC* A.mU.mC. 3626 P.mU.fU. A. A.fC. 27% A. G.mU. A.fC.fU. G. A.fU. G. A* G.mU.mU*mA*mA. A*fC*fC* A* A* G. TEG-Chl 21729 2081 3627 mU*mC*mA.mU.mC. 3628 P.mU.fU. A. A.fC. 30% mA.mG.mU.mG. A.fC.fU. G. A.fU. G. A* mU.mU*mA*mA.TEG- A*fC*fC* A* A* G. Chl 21375 2081 3629 mU.mC. A.mU.mC. 3630 P.mU.fU. A. A.fC. 29% A. G.mU. A.fC.fU. G. A.fU. G. A* G.mU.mU*mA*mA. A*mC*mC* A* A* G. TEG-Chl 21376 2081 3631 mU.mC. A.mU.mC. 3632 P.mU.fU. A. A.fC. 30% A. G.mU. A.fC.fU. G. A.fU. G. A* G.mU.mU*mA*mA. A*fC*fC*mA*mA* G. TEG-Chl 21377 2081 3633 mU.mC. A.mU.mC. 3634 P.mU.fU. A. A.fC. 37% A. G.mU. A.fC.fU. G. A.fU. G.mU.mU*mA*mA. G.mA*mA*fC*fC*mA* TEG-Chl mA* G. 21378 2081 3635 mU*mC*mA.mU.mC. 3636 P.mU.fU. A. A.fC. 32% mA.mG.mU.mG. A.fC.fU. G. A.fU. G. A* mU.mU*mA*mA.TEG- A*mC*mC* A* A* G. Chl 21379 2081 3637 mU*mC*mA.mU.mC. 3638 P.mU.fU. A. A.fC. 31% mA.mG.mU.mG. A.fC.fU. G. A.fU. G. A* mU.mU*mA*mA.TEG- A*fC*fC*mA*mA* G. Chl 21380 2081 3639 mU*mC*mA.mU.mC. 3640 P.mU.fU. A. A.fC. 39% mA.mG.mU.mG. A.fC.fU. G. A.fU. mU.mU*mA*mA.TEG- G.mA*mA*fC*fC*mA* Chl mA* G.

TABLE 22 Inhibition of gene expression with TGFB1 sd-rxRNA sequences (Accession Number: NM_000660.3) % remaining Oligo Start SEQ ID SEQ ID expression Number Site NO Sense sequence NO Antisense sequence (1 uM A549) 14394 1194 3641 G.mC.mU. A. A.mU. 3642 P.mU.fU.fC.fC. A.fC.fC. 24% G. G.mU. G. G. A. A.fU.fU. A. G.mC* A.Chl A*mC* G*mC* G* G. 14395 2006 3643 mU. G. A.mU.mC. 3644 P.mG. A. G.fC. G.fC. 79% G.mU. G.mC. A.fC. G. A.mU.mC. G.mC.mU.mC.Chl A*mU* G*mU*mU* G* G. 14396 1389 3645 mC. A. 3646 P.mU.fC. G.fC.fC. A. G. 77% A.mU.mU.mC.mC.mU. G. A. A.mU.mU. G. G.mC. G. A.Chl G*mU*mU* G*mC*mU* G. 14397 1787 3647 A. G.mU. G. G. 3648 P.mU.fC. G.fU. G. G. n/a A.mU.mC.mC. A.mC. A.fU.fC.fC. G. A.Chl A.mC.mU*mU*mC*mC* A* G* C. 14398 1867 3649 mU. A.mC. A. G.mC. 3650 P.mG. G. A.fC.fC.fU.fU. 82% A. A. G. G.fC.fU. G.mU. G.mU.mC.mC.Chl A*mC*mU* G*mC* G* U. 14399 2002 3651 A. A.mC. A.mU. G. 3652 P.mG.fC. A.fC. G. n/a A.mU.mC. G.mU. A.fU.fC. A.fU. G.mC.Chl G.mU.mU* G* G* A*mC* A* G. 14400 2003 3653 A.mC. A.mU. G. 3654 P.mC. G.fC. A.fC. G. n/a A.mU.mC. G.mU. A.fU.fC. A.mU. G.mC. G.Chl G.mU*mU* G* G* A*mC* A. 14401 1869 3655 mC. A. G.mC. A. A. G. 3656 P.mC. A. G. G. 82% G.mU.mC.mC.mU. A.fC.fC.fU.fU. G.Chl G.mC.mU. G*mU* A*mC*mU* G* C. 14402 2000 3657 mC.mC. A. A.mC. 3658 P.mA.fC. G. A.fU.fC. 66% A.mU. G. A.mU.mC. A.fU. G.fU.mU. G. G* G.mU.Chl A*mC* A* G*mC* U. 14403 986 3659 A. G.mC. G. G. A. A. 3660 P.mA.fU. G.fC. 78% G.mC. G.mC. G.fC.fU.fU.fC.fC. A.mU.Chl G.mC.mU*mU*mC* A*mC*mC* A. 14404 995 3661 G.mC. A.mU.mC. G. 3662 P.mA.fU. G. 79% A. G. G.mC.mC. G.fC.fC.fU.fC. G. A.mU. A.mU.Chl G.mC* G*mC*mU*mU*mC* C. 14405 963 3663 G. A.mC.mU. 3664 P.mC. A.fU. G.fU.fC. G. 80% A.mU.mC. G. A.mC. A.fU. A. A.mU. G.Chl G.mU.mC*mU*mU* G*mC* A* G. 14406 955 3665 A.mC.mC.mU. G.mC. 3666 P.mU. A. G.fU.fC.fU.fU. 88% A. A. G. A.mC.mU. G.fC. A. G. G.mU* G* A.Chl G* A*mU* A* G. 14407 1721 3667 G.mC.mU.mC.mC. 3668 P.mU.fU.fC.fU.fC.fC. n/a A.mC. G. G. A. G. A. G.fU. G. G. A. A.Chl G.mC*mU* G* A* A* G* C. 18454 1246 3669 mC. A.mC. A. G.mC. 3670 P.mU. A.fU. A.fU. A.fU. 58% A.mU. A.mU. A.mU. G.fC.fU. G.fU. G*fU* A.Chl G*fU* A*fC* U. 18455 1248 3671 mC. A. G.mC. A.mU. 3672 P.mU. A.fU. A.fU. A.fU. 87% A.mU. A.mU. A.mU. A.fU. G.fC.fU. G*fU* A.Chl G*fU* G*fU* A. 18456 1755 3673 G.mU. A.mC. 3674 P.mU. A. A. G.fU.fC. A. 107%  A.mU.mU. G. A.fU. G.fU. A.fC* A* A.mC.mU.mU. A.Chl G*fC*fU* G* C. 18457 1755 3675 mU. G.mU. A.mC. 3676 P.mU. A. A. G.fU.fC. A. 77% A.mU.mU. G. A.fU. G.fU. A.fC* A* A.mC.mU.mU. A.Chl G*fC*fU* G* C. 18458 1708 3677 A. A.mC.mU. 3678 P.mU. G. A. A. G.fC. A. 75% A.mU.mU. A.fU. A. G.fU.fU* G* G.mC.mU.mU.mC. G*fU* G*fU* C. A.Chl 18459 1708 3679 mC. A. A.mC.mU. 3680 P.mU. G. A. A. G.fC. A. 73% A.mU.mU. A.fU. A. G.fU.fU* G* G.mC.mU.mU.mC. G*fU* G*fU* C. A.Chl 18460 1250 3681 G.mC. A.mU. A.mU. 3682 P.mU. A.fC. A.fU. A.fU. n/a A.mU. A.mU. G.mU. A.fU. A.fU. G.fC*fU* A.Chl G*fU* G*fU* G. 18461 1754 3683 mU. G.mU. A.mC. 3684 P.mU. A. G.fU.fC. A. 91% A.mU.mU. G. A.fU. G.fU. A.fC. A* A.mC.mU. A.Chl G*fC*fU* G*fC* C. 18462 1754 3685 mC.mU. G.mU. 3686 P.mU. A. G.fU.fC. A. 92% A.mC. A.mU.mU. G. A.fU. G.fU. A.fC. A* A.mC.mU. A.Chl G*fC*fU* G*fC* C. 18463 1249 3687 A. G.mC. A.mU. 3688 P.mU.fC. A.fU. A.fU. n/a A.mU. A.mU. A.mU. A.fU. A.fU. G.fC.fU* G. A.Chl G*fU* G*fU* G* U. 18464 1383 3689 mC. A. G.mC. A. 3690 P.mU. G. A. A.fU.fU. 77% A.mC. A. G.fU.fU. G.fC.fU. G*fU* A.mU.mU.mC. A.Chl A*fU*fU*fU* C. 18465 1251 3691 mC. A.mU. A.mU. 3692 P.mU. A. A.fC. A.fU. 84% A.mU. A.mU. A.fU. A.fU. A.fU. G.mU.mU. A.Chl G*fC*fU* G*fU* G* U. 18466 1713 3693 mU.mU. 3694 P.mU. G. A. G.fC.fU. G. n/a G.mC.mU.mU.mC. A. A. A. G.fC. A. A*fU* A* G.mC.mU.mC. A.Chl G*fU*fU* G. 18467 1713 3695 A.mU.mU. 3696 P.mU. G. A. G.fC.fU. G. 83% G.mC.mU.mU.mC. A. A. A. G.fC. A. A*fU* A* G.mC.mU.mC. A.Chl G*fU*fU* G. 18468 1247 3697 A.mC. A. G.mC. 3698 P.mU.fU. A.fU. A.fU. 96% A.mU. A.mU. A.mU. A.fU. G.fC.fU. G.fU* A. A.Chl G*fU* G*fU* A* C. 18469 1712 3699 A.mU.mU. 3700 P.mU. A. G.fC.fU. G. A. 90% G.mC.mU.mU.mC. A. A. G.fC. A. A.fU* A* G.mC.mU. A.Chl G*fU*fU* G* G. 18470 1712 3701 mU. A.mU.mU. 3702 P.mU. A. G.fC.fU. G. A. 98% G.mC.mU.mU.mC. A. A. G.fC. A. A.fU* A* G.mC.mU. A.Chl G*fU*fU* G* G. 18471 1212 3703 mC. A. A. 3704 P.mU.fU. G.fC.fU.fU. G. n/a G.mU.mU.mC. A. A. A. A.fC.fU.fU. G*fU*fC* G.mC. A. A.Chl A*fU* A* G. 18472 1222 3705 mC. A. G. A. G.mU. 3706 P.mU. G.fU. G.fU. G.fU. 45% A.mC. A.mC. A.mC. A.fC.fU.fC.fU. G* A.Chl C*fU*fU* G* A* A. 18473 1228 3707 A.mC. A.mC. A.mC. 3708 P.mU.fU. A.fU. G.fC.fU. 36% A. G.mC. A.mU. A. G.fU. G.fU. G.fU* A.Chl A*fC*fU*fC*fU* G. 18474 1233 3709 mC. A. G.mC. A.mU. 3710 P.mU. A.fU. A.fU. A.fU. 68% A.mU. A.mU. A.mU. A.fU. G.fC.fU. G*fU* A.Chl G*fU* G*fU* A. 18475 1218 3711 mU.mC. A. A. G.mC. 3712 P.mU.fU. A.fC.fU.fC.fU. 64% A. G. A. G.mU. A. G.fC.fU.fU. G. A* A.Chl A*fC*fU*fU* G* U. 18476 1235 3713 A. G.mC. A.mU. 3714 P.mU.fC. A.fU. A.fU. 78% A.mU. A.mU. A.mU. A.fU. A.fU. G.fC.fU* G. A.Chl G*fU* G*fU* G* U. 18477 1225 3715 A. G. A. G.mU. A.mC. 3716 P.mU.fU. G.fU. G.fU. 92% A.mC. A.mC. A. A.Chl G.fU. A.fC.fU.fC.fU* G*fC*fU*fU* G* A. 18478 1221 3717 A. A. G.mC. A. G. A. 3718 P.mU.fU. G.fU. 103%  G.mU. A.mC. A. A.Chl A.fC.fU.fC.fU. G.fC.fU.fU* G* A* A*fC*fU* U. 18479 1244 3719 mU.mU.mC. A. A.mC. 3720 P.mU.fU. G. A.fU. G.fU. 84% A.mC. A.mU.mC. A. G.fU.fU. G. A. A* G* A* A.Chl A*fC* A* U. 18480 1224 3721 A. G.mC. A. G. A. 3722 P.mU. G.fU. G.fU. 37% G.mU. A.mC. A.mC. A.fC.fU.fC.fU. A.Chl G.fC.fU*fU* G* A* A*fC* U. 18481 1242 3723 A.mU. A.mU. A.mU. 3724 P.mU. A. A. G. A. A.fC. 62% G.mU.mU.mC.mU.mU. A.fU. A.fU. A.fU* A*fU* A.Chl G*fC*fU* G. 18482 1213 3725 G. A.mC. A. A. 3726 P.mU.fC.fU.fU. G. A. 47% G.mU.mU.mC. A. A. A.fC.fU.fU. G.fU.fC* G. A.Chl A*fU* A* G* A* U. 18483 1760 3727 mU.mU. A. A. A. G. 3728 P.mU.fC.fU.fC.fC. 69% A.mU. G. G. A. G. A.fU.fC.fU.fU.fU. A. A.Chl A*fU* G* G* G* G* C. 18484 1211 3729 mC.mU. A.mU. G. 3730 P.mU. A. A.fC.fU.fU. n/a A.mC. A. A. G.fU.fC. A.fU. A. G* G.mU.mU. A.Chl A*fU*fU*fU*fC* G. 19411 1212 3731 mC. A. A.mC. G. A. A. 3732 P.mU.fU. A. G. 52% A.mU.mC.mU. A. A.fU.fU.fU.fC. G.fU.fU. A.Chl G*fU* G* G* G*fU*fU. 19412 1222 3733 mU. A.mU. G. A.mC. 3734 P.mU. G. A. A.fC.fU.fU. 51% A. A. G.mU.mU.mC. G.fU.fC. A.fU. A* G* A.Chl A*fU*fU*fU*fC. 19413 1228 3735 A. A. G.mU.mU.mC. 3736 P.mU.fC.fU. G.fC.fU.fU. n/a A. A. G.mC. A. G. G. A. A.fC.fU.fU* A.Chl G*fU*fC* A*fU* A. 19414 1233 3737 mC. A. A. G.mC. A. G. 3738 P.mU. G.fU. 41% A. G.mU. A.mC. A.Chl A.fC.fU.fC.fU. G.fC.fU.fU. G* A* A*fC*fU*fU* G. 19415 1218 3739 A. A.mU.mC.mU. 3740 P.mU.fU.fU. G.fU.fC. 104%  A.mU. G. A.mC. A. A. A.fU. A. G. A.Chl A.fU.fU*fU*fC* G*fU*fU* G. 19416 1244 3741 mC. A.mC. A.mC. A. 3742 P.mU. A.fU. A.fU. 31% G.mC. A.mU. A.mU. G.fC.fU. G.fU. G.fU. A.Chl G*fU* A*fC*fU*fC*fU. 19417 655 3743 G. A. A. A.mU. 3744 P.mU.fU.fU. G.fC.fU. n/a A.mU. A. G.mC. A. A. A.fU. A.fU.fU.fU.fC*fU* A.Chl G* G*fU* A* G. 19418 644 3745 G. A. 3746 P.mU.fC.fU. G. G.fU. A. n/a A.mC.mU.mC.mU. G. A. G.fU.fU.fC*fU* A.mC.mC. A. G. A.Chl A*fC* G*fU* G. 19419 819 3747 G.mC. A. A. A. G. 3748 P.mU.fC. A.fU.fU. n/a A.mU. A. A.mU. G. A.fU.fC.fU.fU.fU. A.Chl G.fC*fU* G*fU*fC* A* C. 19420 645 3749 A. A.mC.mU.mC.mU. 3750 P.mU.fU.fC.fU. G. G.fU. n/a A.mC.mC. A. G. A. A. G. A. G.fU.fU*fC*fU* A.Chl A*fC* G* U. 19421 646 3751 A.mC.mU.mC.mU. 3752 P.mU.fU.fU.fC.fU. G. n/a A.mC.mC. A. G. A. A. G.fU. A. G. A. A.Chl G.fU*fU*fC*fU* A*fC* G. 19422 816 3753 A.mC. A. G.mC. A. A. 3754 P.mU.fU. n/a A. G. A.mU. A. A.Chl A.fU.fC.fU.fU.fU. G.fC.fU. G.fU*fC* A*fC* A* A* G. 19423 495 3755 mC. A. A.mU.mC.mU. 3756 P.mU.fU. G.fU.fC. A.fU. n/a A.mU. G. A.mC. A. A. G. A.fU.fU. G*fC* A.Chl G*fU*fU* G* U. 19424 614 3757 A. G. A.mU.mU.mC. 3758 P.mU.fU. G. A.fC.fU.fU. n/a A. A. G.mU.mC. A. G. A. A.fU.fC.fU*fC*fU* A.Chl G*fC* A* G. 19425 627 3759 mC.mU. G.mU. G. G. 3760 P.mU. G.fU.fU. n/a A. G.mC. A. A.mC. G.fC.fU.fC.fC. A.fC. A. A.Chl G*fU*fU* G* A*fC* U. 19426 814 3761 mU. G. A.mC. A. 3762 P.mU.fU.fC.fU.fU.fU. n/a G.mC. A. A. A. G. A. G.fC.fU. G.fU.fC. A*fC* A.Chl A* A* G* A* G. 19427 501 3763 A.mU. G. A.mC. A. A. 3764 P.mU.fU. G. n/a A. A.mC.mC. A. A.Chl G.fU.fU.fU.fU. G.fU.fC. A.fU* A* G* A*fU*fU* G. 19428 613 3765 G. A. G. 3766 P.mU. G. A.fC.fU.fU. G. n/a A.mU.mU.mC. A. A. A. A.fU.fC.fU.fC*fU* G.mU.mC. A.Chl G*fC* A* G* G. 21240 1244 3767 mC. A.mC. A.mC. A. 3768 P.mU. A.fU. A.fU. 0.875 G.mC. A.mU. A.mU. G.fC.fU. G.fU. G.fU. A.Chl G*mU* A*mC*mU*mC* U. 21241 1244 3769 mC. A.mC. A.mC. A. 3770 P.mU. A.fU. A.fU. 0.88  G.mC. A.mU. A.mU. G.fC.fU. G.fU. G.fU. A.Chl G*mU*mA*mC*mU*mC* U. 21242 1244 3771 mC. A.mC. A.mC. A. 3772 P.mU. A.fU. A.fU. 0.635 G.mC. A.mU. A.mU. G.fC.fU. G.fU. A.Chl G.fU.mG*mU*mA*mC* mU*mC* U. 21243 1244 3773 mC. A.mC. A.mC. A. 3774 P.mU. A.fU. A.fU. 0.32  G.mC. A.mU. A.mU. G.fC.fU. G.fU. A.Chl G.fU.mG*fU*mA*fC*mU* fC* U. 21244 1244 3775 mC. A.mC. A.mC. A. 3776 P.mU. A.fU. A.fU. 0.36  G.mC. A.mU. A.mU. G.fC.fU. G.fU. G.fU. A.Chl G*fU* A*fC*mU*mC* U. 21245 1244 3777 mC. A.mC. A.mC. A. 3778 P.mU. A.fU. A.fU. 0.265 G.mC. A.mU. G.fC.fU. G.fU. G.fU. A*mU*mA.TEG-Chl G*fU* A*fC*fU*fC*fU. 21246 1244 3779 mC*mA*mC. A.mC. 3780 P.mU. A.fU. A.fU. 0.334 A. G.mC. A.mU. G.fC.fU. G.fU. G.fU. A*mU*mA.TEG-Chl G*fU* A*fC*fU*fC*fU. 21247 1244 3781 mC*mA*mC.mA.mC. 3782 P.mU. A.fU. A.fU. 0.29  mA.mG.mC.mA.mU. G.fC.fU. G.fU. G.fU. mA*mU*mA.TEG-Chl G*fU* A*fC*fU*fC*fU. 21248 614 3783 mA. G. 3784 P.mU.fU. G. A.fC.fU.fU. n/a A.mU.mU.mC. A. A. G. A. A.fU.fC.fU*fC*fU* G.mU.mC*mA*mA.TEG- G*fC*fU* U. Chl 20608 1244 3785 mC. A.mC. A.mC. A. 3786 P.mU. A.fU. A.fU. 79% G.mC. A.mU. A.mU. G.fC.fU. G.fU. G.mU. A.Chl G*mU* A*mC*mU*mC* U. 20609 1244 3787 mC. A.mC. A.mC. A. 3788 P.mU. A.fU. A.fU. 60% G.mC. A.mU. A.mU. G.fC.fU. G.fU. G.mU. A.Chl G*fU* A*mC*fU*mC* U. 20610 1244 3789 mC. A.mC. A.mC. A. 3790 P.mU. A. U. A. U. G. C. 93% G.mC. A.mU. A.mU. U. G. U. G.mU. G*mU* A.Chl A*mC*mU*mC* U. 20611 1244 3791 mC. A.mC. A.mC. A. 3792 P.mU. A.fU. A.fU. n/a G.mC. A.mU. A.mU. G.fC.fU. G.fU. A.Chl G.mU.mG*mU*mA*mC* mU*mC* U. 21374 614 3793 mC*mA*mC.mA.mC. 3794 P.mU. A.fU. A.fU. 24% mA.mG.mC.mA.mU. G.fC.fU. G.fU. mA*mU*mA.TEG-Chl G.fU.mG*fU*mA*fC*mU* fC* U.

TABLE 23 CB1 sequences Ref SEQ ID Pos SEQ ID NO 19-mer Sense Seq NO 25-mer Sense Seq w/A @ 25 1690 3795 AUGUCUGUGUCCACAGACA 3796 GUAACCAUGUCUGUGUCCACAGACA 1686 3797 AACCAUGUCUGUGUCCACA 3798 CAAGGUAACCAUGUCUGUGUCCACA 1685 3799 UAACCAUGUCUGUGUCCAC 3800 CCAAGGUAACCAUGUCUGUGUCCAA 1684 3801 GUAACCAUGUCUGUGUCCA 3802 GCCAAGGUAACCAUGUCUGUGUCCA 1649 3803 AAAGCUGCAUCAAGAGCAC 3804 CCGCAGAAAGCUGCAUCAAGAGCAA 1648 3805 GAAAGCUGCAUCAAGAGCA 3806 GCCGCAGAAAGCUGCAUCAAGAGCA 1494 3807 CAUCUAUGCUCUGAGGAGU 3808 CCCCAUCAUCUAUGCUCUGAGGAGA 1493 3809 UCAUCUAUGCUCUGAGGAG 3810 ACCCCAUCAUCUAUGCUCUGAGGAA 1492 3811 AUCAUCUAUGCUCUGAGGA 3812 AACCCCAUCAUCUAUGCUCUGAGGA 1491 3813 CAUCAUCUAUGCUCUGAGG 3814 GAACCCCAUCAUCUAUGCUCUGAGA 1490 3815 CCAUCAUCUAUGCUCUGAG 3816 UGAACCCCAUCAUCUAUGCUCUGAA 1489 3817 CCCAUCAUCUAUGCUCUGA 3818 GUGAACCCCAUCAUCUAUGCUCUGA 1487 3819 ACCCCAUCAUCUAUGCUCU 3820 CCGUGAACCCCAUCAUCUAUGCUCA 1486 3821 AACCCCAUCAUCUAUGCUC 3822 ACCGUGAACCCCAUCAUCUAUGCUA 1358 3823 UGGUGUUGAUCAUCUGCUG 3824 UCCUGGUGGUGUUGAUCAUCUGCUA 1357 3825 GUGGUGUUGAUCAUCUGCU 3826 AUCCUGGUGGUGUUGAUCAUCUGCA 1355 3827 UGGUGGUGUUGAUCAUCUG 3828 UGAUCCUGGUGGUGUUGAUCAUCUA 1354 3829 CUGGUGGUGUUGAUCAUCU 3830 CUGAUCCUGGUGGUGUUGAUCAUCA 1351 3831 AUCCUGGUGGUGUUGAUCA 3832 GUCCUGAUCCUGGUGGUGUUGAUCA 1198 3833 AUUCUCUGGAAGGCUCACA 3834 AUGUAUAUUCUCUGGAAGGCUCACA 1197 3835 UAUUCUCUGGAAGGCUCAC 3836 CAUGUAUAUUCUCUGGAAGGCUCAA 1196 3837 AUAUUCUCUGGAAGGCUCA 3838 ACAUGUAUAUUCUCUGGAAGGCUCA 1195 3839 UAUAUUCUCUGGAAGGCUC 3840 UACAUGUAUAUUCUCUGGAAGGCUA 1131 3841 CUACCUGAUGUUCUGGAUC 3842 UGAAACCUACCUGAUGUUCUGGAUA 1129 3843 ACCUACCUGAUGUUCUGGA 3844 GAUGAAACCUACCUGAUGUUCUGGA 1127 3845 AAACCUACCUGAUGUUCUG 3846 UUGAUGAAACCUACCUGAUGUUCUA 1126 3847 GAAACCUACCUGAUGUUCU 3848 AUUGAUGAAACCUACCUGAUGUUCA 1086 3849 ACUGCAAUCUGUUUGCUCA 3850 CGAGAAACUGCAAUCUGUUUGCUCA 1084 3851 AAACUGCAAUCUGUUUGCU 3852 UGCGAGAAACUGCAAUCUGUUUGCA 972 3853 CCUGGCCUAUAAGAGGAUU 3854 CAGGCCCCUGGCCUAUAAGAGGAUA 951 3855 GUACAUAUCCAUUCACAGG 3856 CGACAGGUACAUAUCCAUUCACAGA 950 3857 GGUACAUAUCCAUUCACAG 3858 UCGACAGGUACAUAUCCAUUCACAA 948 3859 CAGGUACAUAUCCAUUCAC 3860 CAUCGACAGGUACAUAUCCAUUCAA 947 3861 ACAGGUACAUAUCCAUUCA 3862 CCAUCGACAGGUACAUAUCCAUUCA 946 3863 GACAGGUACAUAUCCAUUC 3864 GCCAUCGACAGGUACAUAUCCAUUA 943 3865 AUCGACAGGUACAUAUCCA 3866 ACAGCCAUCGACAGGUACAUAUCCA 941 3867 CCAUCGACAGGUACAUAUC 3868 UCACAGCCAUCGACAGGUACAUAUA 940 3869 GCCAUCGACAGGUACAUAU 3870 CUCACAGCCAUCGACAGGUACAUAA 869 3871 ACGUGUUUCUGUUCAAACU 3872 GCCGCAACGUGUUUCUGUUCAAACA 868 3873 AACGUGUUUCUGUUCAAAC 3874 AGCCGCAACGUGUUUCUGUUCAAAA 1647 3875 AGAAAGCUGCAUCAAGAGC 3876 GGCCGCAGAAAGCUGCAUCAAGAGA 1645 3877 GCAGAAAGCUGCAUCAAGA 3878 AGGGCCGCAGAAAGCUGCAUCAAGA 1394 3879 UCAUGGUGUAUGAUGUCUU 3880 UUGCAAUCAUGGUGUAUGAUGUCUA 1393 3881 AUCAUGGUGUAUGAUGUCU 3882 CUUGCAAUCAUGGUGUAUGAUGUCA 1391 3883 CAAUCAUGGUGUAUGAUGU 3884 UGCUUGCAAUCAUGGUGUAUGAUGA 1125 3885 UGAAACCUACCUGAUGUUC 3886 CAUUGAUGAAACCUACCUGAUGUUA 1090 3887 CAAUCUGUUUGCUCAGACA 3888 AAACUGCAAUCUGUUUGCUCAGACA 1089 3889 GCAAUCUGUUUGCUCAGAC 3890 GAAACUGCAAUCUGUUUGCUCAGAA 1088 3891 UGCAAUCUGUUUGCUCAGA 3892 AGAAACUGCAAUCUGUUUGCUCAGA 1087 3893 CUGCAAUCUGUUUGCUCAG 3894 GAGAAACUGCAAUCUGUUUGCUCAA 1397 3895 UGGUGUAUGAUGUCUUUGG 3896 CAAUCAUGGUGUAUGAUGUCUUUGA 1396 3897 AUGGUGUAUGAUGUCUUUG 3898 GCAAUCAUGGUGUAUGAUGUCUUUA 1120 3899 AUUGAUGAAACCUACCUGA 3900 CCACACAUUGAUGAAACCUACCUGA 1118 3901 ACAUUGAUGAAACCUACCU 3902 UCCCACACAUUGAUGAAACCUACCA 1117 3903 CACAUUGAUGAAACCUACC 3904 UUCCCACACAUUGAUGAAACCUACA 1116 3905 ACACAUUGAUGAAACCUAC 3906 UUUCCCACACAUUGAUGAAACCUAA 1132 3907 UACCUGAUGUUCUGGAUCG 3908 GAAACCUACCUGAUGUUCUGGAUCA 845 3909 UGUUCCACCGCAAAGAUAG 3910 UCCACGUGUUCCACCGCAAAGAUAA 844 3911 GUGUUCCACCGCAAAGAUA 3912 UUCCACGUGUUCCACCGCAAAGAUA 573 3913 CUUCAAGGAGAAUGAGGAG 3914 CUCGUCCUUCAAGGAGAAUGAGGAA 572 3915 CCUUCAAGGAGAAUGAGGA 3916 UCUCGUCCUUCAAGGAGAAUGAGGA 571 3917 UCCUUCAAGGAGAAUGAGG 3918 CUCUCGUCCUUCAAGGAGAAUGAGA 1449 3919 AUUCUGCAGUAUGCUCUGC 3920 GUUUGCAUUCUGCAGUAUGCUCUGA 1448 3921 CAUUCUGCAGUAUGCUCUG 3922 UGUUUGCAUUCUGCAGUAUGCUCUA 1447 3923 GCAUUCUGCAGUAUGCUCU 3924 GUGUUUGCAUUCUGCAGUAUGCUCA 1253 3925 AGAGCAUCAUCAUCCACAC 3926 CCCAGAAGAGCAUCAUCAUCCACAA 1252 3927 AAGAGCAUCAUCAUCCACA 3928 ACCCAGAAGAGCAUCAUCAUCCACA 1247 3929 CCCAGAAGAGCAUCAUCAU 3930 GUGGCACCCAGAAGAGCAUCAUCAA 1246 3931 ACCCAGAAGAGCAUCAUCA 3932 CGUGGCACCCAGAAGAGCAUCAUCA 311 3933 UGAAGUCGAUCCUAGAUGG 3934 AGGUUAUGAAGUCGAUCCUAGAUGA 310 3935 AUGAAGUCGAUCCUAGAUG 3936 GAGGUUAUGAAGUCGAUCCUAGAUA 1249 3937 CAGAAGAGCAUCAUCAUCC 3938 GGCACCCAGAAGAGCAUCAUCAUCA 585 3939 UGAGGAGAACAUCCAGUGU 3940 GGAGAAUGAGGAGAACAUCCAGUGA 583 3941 AAUGAGGAGAACAUCCAGU 3942 AAGGAGAAUGAGGAGAACAUCCAGA 581 3943 AGAAUGAGGAGAACAUCCA 3944 UCAAGGAGAAUGAGGAGAACAUCCA 580 3945 GAGAAUGAGGAGAACAUCC 3946 UUCAAGGAGAAUGAGGAGAACAUCA 579 3947 GGAGAAUGAGGAGAACAUC 3948 CUUCAAGGAGAAUGAGGAGAACAUA 578 3949 AGGAGAAUGAGGAGAACAU 3950 CCUUCAAGGAGAAUGAGGAGAACAA 577 3951 AAGGAGAAUGAGGAGAACA 3952 UCCUUCAAGGAGAAUGAGGAGAACA 574 3953 UUCAAGGAGAAUGAGGAGA 3954 UCGUCCUUCAAGGAGAAUGAGGAGA 1257 3955 CAUCAUCAUCCACACGUCU 3956 GAAGAGCAUCAUCAUCCACACGUCA 1255 3957 AGCAUCAUCAUCCACACGU 3958 CAGAAGAGCAUCAUCAUCCACACGA 1682 3959 AGGUAACCAUGUCUGUGUC 3960 UUGCCAAGGUAACCAUGUCUGUGUA 1681 3961 AAGGUAACCAUGUCUGUGU 3962 AUUGCCAAGGUAACCAUGUCUGUGA 1680 3963 CAAGGUAACCAUGUCUGUG 3964 GAUUGCCAAGGUAACCAUGUCUGUA 1499 3965 AUGCUCUGAGGAGUAAGGA 3966 UCAUCUAUGCUCUGAGGAGUAAGGA 1498 3967 UAUGCUCUGAGGAGUAAGG 3968 AUCAUCUAUGCUCUGAGGAGUAAGA 1497 3969 CUAUGCUCUGAGGAGUAAG 3970 CAUCAUCUAUGCUCUGAGGAGUAAA 1496 3971 UCUAUGCUCUGAGGAGUAA 3972 CCAUCAUCUAUGCUCUGAGGAGUAA 1388 3973 UUGCAAUCAUGGUGUAUGA 3974 CUCUGCUUGCAAUCAUGGUGUAUGA 1387 3975 CUUGCAAUCAUGGUGUAUG 3976 CCUCUGCUUGCAAUCAUGGUGUAUA 1386 3977 GCUUGCAAUCAUGGUGUAU 3978 CCCUCUGCUUGCAAUCAUGGUGUAA 1385 3979 UGCUUGCAAUCAUGGUGUA 3980 GCCCUCUGCUUGCAAUCAUGGUGUA 1384 3981 CUGCUUGCAAUCAUGGUGU 3982 GGCCCUCUGCUUGCAAUCAUGGUGA 1383 3983 UCUGCUUGCAAUCAUGGUG 3984 GGGCCCUCUGCUUGCAAUCAUGGUA 1382 3985 CUCUGCUUGCAAUCAUGGU 3986 GGGGCCCUCUGCUUGCAAUCAUGGA 1314 3987 CCGCAUGGACAUUAGGUUA 3988 CCAAGCCCGCAUGGACAUUAGGUUA 1094 3989 CUGUUUGCUCAGACAUUUU 3990 UGCAAUCUGUUUGCUCAGACAUUUA 1093 3991 UCUGUUUGCUCAGACAUUU 3992 CUGCAAUCUGUUUGCUCAGACAUUA 1083 3993 GAAACUGCAAUCUGUUUGC 3994 CUGCGAGAAACUGCAAUCUGUUUGA 1082 3995 AGAAACUGCAAUCUGUUUG 3996 ACUGCGAGAAACUGCAAUCUGUUUA 1080 3997 CGAGAAACUGCAAUCUGUU 3998 GAACUGCGAGAAACUGCAAUCUGUA 323 3999 UAGAUGGCCUUGCAGAUAC 4000 CGAUCCUAGAUGGCCUUGCAGAUAA 322 4001 CUAGAUGGCCUUGCAGAUA 4002 UCGAUCCUAGAUGGCCUUGCAGAUA 1179 4003 CGUGUAUGCGUACAUGUAU 4004 GUUCAUCGUGUAUGCGUACAUGUAA 1178 4005 UCGUGUAUGCGUACAUGUA 4006 UGUUCAUCGUGUAUGCGUACAUGUA 1177 4007 AUCGUGUAUGCGUACAUGU 4008 CUGUUCAUCGUGUAUGCGUACAUGA 1320 4009 GGACAUUAGGUUAGCCAAG 4010 CCGCAUGGACAUUAGGUUAGCCAAA 1319 4011 UGGACAUUAGGUUAGCCAA 4012 CCCGCAUGGACAUUAGGUUAGCCAA 1318 4013 AUGGACAUUAGGUUAGCCA 4014 GCCCGCAUGGACAUUAGGUUAGCCA 1317 4015 CAUGGACAUUAGGUUAGCC 4016 AGCCCGCAUGGACAUUAGGUUAGCA 1316 4017 GCAUGGACAUUAGGUUAGC 4018 AAGCCCGCAUGGACAUUAGGUUAGA 1315 4019 CGCAUGGACAUUAGGUUAG 4020 CAAGCCCGCAUGGACAUUAGGUUAA 1415 4021 GGAAGAUGAACAAGCUCAU 4022 UCUUUGGGAAGAUGAACAAGCUCAA 552 4023 UUACAACAAGUCUCUCUCG 4024 AGAAUUUUACAACAAGUCUCUCUCA 551 4025 UUUACAACAAGUCUCUCUC 4026 CAGAAUUUUACAACAAGUCUCUCUA 550 4027 UUUUACAACAAGUCUCUCU 4028 ACAGAAUUUUACAACAAGUCUCUCA 476 4029 GUCCCUUCCAAGAGAAGAU 4030 GGGGAAGUCCCUUCCAAGAGAAGAA 474 4031 AAGUCCCUUCCAAGAGAAG 4032 UAGGGGAAGUCCCUUCCAAGAGAAA 473 4033 GAAGUCCCUUCCAAGAGAA 4034 UUAGGGGAAGUCCCUUCCAAGAGAA 1020 4035 UUGCCUGAUGUGGACCAUA 4036 GGCGUUUUGCCUGAUGUGGACCAUA 1019 4037 UUUGCCUGAUGUGGACCAU 4038 UGGCGUUUUGCCUGAUGUGGACCAA 1018 4039 UUUUGCCUGAUGUGGACCA 4040 GUGGCGUUUUGCCUGAUGUGGACCA 606 4041 GGAGAACUUCAUGGACAUA 4042 GUGUGGGGAGAACUUCAUGGACAUA 1568 4043 AGCCUCUGGAUAACAGCAU 4044 CUGCGCAGCCUCUGGAUAACAGCAA 1170 4045 UCUGUUCAUCGUGUAUGCG 4046 ACUGCUUCUGUUCAUCGUGUAUGCA 1169 4047 UUCUGUUCAUCGUGUAUGC 4048 UACUGCUUCUGUUCAUCGUGUAUGA 1168 4049 CUUCUGUUCAUCGUGUAUG 4050 GUACUGCUUCUGUUCAUCGUGUAUA 1421 4051 UGAACAAGCUCAUUAAGAC 4052 GGAAGAUGAACAAGCUCAUUAAGAA 1420 4053 AUGAACAAGCUCAUUAAGA 4054 GGGAAGAUGAACAAGCUCAUUAAGA 1419 4055 GAUGAACAAGCUCAUUAAG 4056 UGGGAAGAUGAACAAGCUCAUUAAA 1418 4057 AGAUGAACAAGCUCAUUAA 4058 UUGGGAAGAUGAACAAGCUCAUUAA 1417 4059 AAGAUGAACAAGCUCAUUA 4060 UUUGGGAAGAUGAACAAGCUCAUUA 1172 4061 UGUUCAUCGUGUAUGCGUA 4062 UGCUUCUGUUCAUCGUGUAUGCGUA 1078 4063 UGCGAGAAACUGCAAUCUG 4064 UGGAACUGCGAGAAACUGCAAUCUA 825 4065 CAGCUUCAUUGACUUCCAC 4066 UGUCUACAGCUUCAUUGACUUCCAA 824 4067 ACAGCUUCAUUGACUUCCA 4068 UUGUCUACAGCUUCAUUGACUUCCA 823 4069 UACAGCUUCAUUGACUUCC 4070 UUUGUCUACAGCUUCAUUGACUUCA 821 4071 UCUACAGCUUCAUUGACUU 4072 UUUUUGUCUACAGCUUCAUUGACUA 820 4073 GUCUACAGCUUCAUUGACU 4074 AUUUUUGUCUACAGCUUCAUUGACA 612 4075 CUUCAUGGACAUAGAGUGU 4076 GGAGAACUUCAUGGACAUAGAGUGA 611 4077 ACUUCAUGGACAUAGAGUG 4078 GGGAGAACUUCAUGGACAUAGAGUA 610 4079 AACUUCAUGGACAUAGAGU 4080 GGGGAGAACUUCAUGGACAUAGAGA 549 4081 AUUUUACAACAAGUCUCUC 4082 UACAGAAUUUUACAACAAGUCUCUA 547 4083 GAAUUUUACAACAAGUCUC 4084 AUUACAGAAUUUUACAACAAGUCUA 1176 4085 CAUCGUGUAUGCGUACAUG 4086 UCUGUUCAUCGUGUAUGCGUACAUA 1175 4087 UCAUCGUGUAUGCGUACAU 4088 UUCUGUUCAUCGUGUAUGCGUACAA 1174 4089 UUCAUCGUGUAUGCGUACA 4090 CUUCUGUUCAUCGUGUAUGCGUACA 1173 4091 GUUCAUCGUGUAUGCGUAC 4092 GCUUCUGUUCAUCGUGUAUGCGUAA 1171 4093 CUGUUCAUCGUGUAUGCGU 4094 CUGCUUCUGUUCAUCGUGUAUGCGA 609 4095 GAACUUCAUGGACAUAGAG 4096 UGGGGAGAACUUCAUGGACAUAGAA 608 4097 AGAACUUCAUGGACAUAGA 4098 GUGGGGAGAACUUCAUGGACAUAGA 607 4099 GAGAACUUCAUGGACAUAG 4100 UGUGGGGAGAACUUCAUGGACAUAA 1322 4101 ACAUUAGGUUAGCCAAGAC 4102 GCAUGGACAUUAGGUUAGCCAAGAA 1321 4103 GACAUUAGGUUAGCCAAGA 4104 CGCAUGGACAUUAGGUUAGCCAAGA 1027 4105 AUGUGGACCAUAGCCAUUG 4106 UGCCUGAUGUGGACCAUAGCCAUUA 545 4107 CAGAAUUUUACAACAAGUC 4108 ACAUUACAGAAUUUUACAACAAGUA 532 4109 CAGGUGAACAUUACAGAAU 4110 GCAGACCAGGUGAACAUUACAGAAA 813 4111 CAUUUUUGUCUACAGCUUC 4112 GAGUGUCAUUUUUGUCUACAGCUUA 812 4113 UCAUUUUUGUCUACAGCUU 4114 GGAGUGUCAUUUUUGUCUACAGCUA 811 4115 GUCAUUUUUGUCUACAGCU 4116 GGGAGUGUCAUUUUUGUCUACAGCA 809 4117 GUGUCAUUUUUGUCUACAG 4118 UGGGGAGUGUCAUUUUUGUCUACAA 808 4119 AGUGUCAUUUUUGUCUACA 4120 CUGGGGAGUGUCAUUUUUGUCUACA 569 4121 CGUCCUUCAAGGAGAAUGA 4122 CUCUCUCGUCCUUCAAGGAGAAUGA 568 4123 UCGUCCUUCAAGGAGAAUG 4124 UCUCUCUCGUCCUUCAAGGAGAAUA 1444 4125 UUUGCAUUCUGCAGUAUGC 4126 ACGGUGUUUGCAUUCUGCAGUAUGA 1443 4127 GUUUGCAUUCUGCAGUAUG 4128 GACGGUGUUUGCAUUCUGCAGUAUA 1446 4129 UGCAUUCUGCAGUAUGCUC 4130 GGUGUUUGCAUUCUGCAGUAUGCUA 1445 4131 UUGCAUUCUGCAGUAUGCU 4132 CGGUGUUUGCAUUCUGCAGUAUGCA 1442 4133 UGUUUGCAUUCUGCAGUAU 4134 AGACGGUGUUUGCAUUCUGCAGUAA 1677 4135 UGCCAAGGUAACCAUGUCU 4136 CAAGAUUGCCAAGGUAACCAUGUCA 1676 4137 UUGCCAAGGUAACCAUGUC 4138 UCAAGAUUGCCAAGGUAACCAUGUA 1675 4139 AUUGCCAAGGUAACCAUGU 4140 GUCAAGAUUGCCAAGGUAACCAUGA 1603 4141 CUGCACAAACACGCAAACA 4142 GACUGCCUGCACAAACACGCAAACA 1110 4143 UUUCCCACACAUUGAUGAA 4144 AGACAUUUUCCCACACAUUGAUGAA 1109 4145 UUUUCCCACACAUUGAUGA 4146 CAGACAUUUUCCCACACAUUGAUGA 1108 4147 AUUUUCCCACACAUUGAUG 4148 UCAGACAUUUUCCCACACAUUGAUA 1605 4149 GCACAAACACGCAAACAAU 4150 CUGCCUGCACAAACACGCAAACAAA 1604 4151 UGCACAAACACGCAAACAA 4152 ACUGCCUGCACAAACACGCAAACAA 1671 4153 CAAGAUUGCCAAGGUAACC 4154 CACGGUCAAGAUUGCCAAGGUAACA 1670 4155 UCAAGAUUGCCAAGGUAAC 4156 GCACGGUCAAGAUUGCCAAGGUAAA 1669 4157 GUCAAGAUUGCCAAGGUAA 4158 AGCACGGUCAAGAUUGCCAAGGUAA 628 4159 UGUUUCAUGGUCCUGAACC 4160 AUAGAGUGUUUCAUGGUCCUGAACA 1115 4161 CACACAUUGAUGAAACCUA 4162 UUUUCCCACACAUUGAUGAAACCUA 1114 4163 CCACACAUUGAUGAAACCU 4164 AUUUUCCCACACAUUGAUGAAACCA 1113 4165 CCCACACAUUGAUGAAACC 4166 CAUUUUCCCACACAUUGAUGAAACA 1112 4167 UCCCACACAUUGAUGAAAC 4168 ACAUUUUCCCACACAUUGAUGAAAA 1111 4169 UUCCCACACAUUGAUGAAA 4170 GACAUUUUCCCACACAUUGAUGAAA 814 4171 AUUUUUGUCUACAGCUUCA 4172 AGUGUCAUUUUUGUCUACAGCUUCA 1659 4173 CAAGAGCACGGUCAAGAUU 4174 CUGCAUCAAGAGCACGGUCAAGAUA 1657 4175 AUCAAGAGCACGGUCAAGA 4176 AGCUGCAUCAAGAGCACGGUCAAGA 1167 4177 GCUUCUGUUCAUCGUGUAU 4178 CGUACUGCUUCUGUUCAUCGUGUAA 1166 4179 UGCUUCUGUUCAUCGUGUA 4180 GCGUACUGCUUCUGUUCAUCGUGUA 1668 4181 GGUCAAGAUUGCCAAGGUA 4182 GAGCACGGUCAAGAUUGCCAAGGUA 819 4183 UGUCUACAGCUUCAUUGAC 4184 CAUUUUUGUCUACAGCUUCAUUGAA 818 4185 UUGUCUACAGCUUCAUUGA 4186 UCAUUUUUGUCUACAGCUUCAUUGA 817 4187 UUUGUCUACAGCUUCAUUG 4188 GUCAUUUUUGUCUACAGCUUCAUUA 816 4189 UUUUGUCUACAGCUUCAUU 4190 UGUCAUUUUUGUCUACAGCUUCAUA 1543 4191 UUUCCCUCUUGUGAAGGCA 4192 AGCAUGUUUCCCUCUUGUGAAGGCA 1660 4193 AAGAGCACGGUCAAGAUUG 4194 UGCAUCAAGAGCACGGUCAAGAUUA 1030 4195 UGGACCAUAGCCAUUGUGA 4196 CUGAUGUGGACCAUAGCCAUUGUGA 531 4197 CCAGGUGAACAUUACAGAA 4198 AGCAGACCAGGUGAACAUUACAGAA 1259 4199 UCAUCAUCCACACGUCUGA 4200 AGAGCAUCAUCAUCCACACGUCUGA 1258 4201 AUCAUCAUCCACACGUCUG 4202 AAGAGCAUCAUCAUCCACACGUCUA Chemical Modification Key Chl cholesterol with hydroxyprolinol linker TEG- cholesterol with TEG linker Chl m 2′Ome f 2′fluoro * phosphorothioate linkage . phosphodiester linkage

TABLE 24 Summary of CTGF Leads Opt. Opt. Generic Targeting lead Opt. lead Name TEG ID 2′F site sequence Optimized lead sequence lead 2′F 2′OH Priority CTGF L1 21045 4 2295 21212 mU.mU. G.mC. A.mC.mC.mU.mU.mU.mC.mU*mA*mA- 4  7(2) 1 chol P.mU.fU. A. G. A. mA. A. G. G.fU. G. fC. mA. mA* mA*fC* mA*mA* mG* G 21214 mU.mU. G.mC. A.mC.mC.mU.mU.mU.mC.mU*mA*mA- 4 12(2) 3 chol P.mU.fU. A. G. A. mA. A. G.fU. G.fC. A. A* A*fC* A*mA*mG*G 21215 mU.mU. G.mC. A. mC.mC.mU.mU.mU.mC.mU*mA*mA- 4 10(2) 2 chol P.mU.fU. A. G. A. mA. A. G. G.fU. G.fC. mA. A* mA*fC* A*mA*mG* G CTGFL2 20393 5 2296 21204 G.mC. A.mC.mC.mU.mU.mU.mC.mU. A*mG*mA-TEG- 3 11(3) 1 Chl P.mU.fC.fU.A.G.mA.A.mA.G.G.fU.G. mC*A*A*A*mC*A*U 21205 G.mC. A.mC.mC.mU.mU.mU.mC.mU. A* 3 10(3) 3 mG*mA-TEG-Chl P.mU.fC.fU.A.G.mA.A.mA. G.G.fU.G.mC*A*mA*A*mC*A*U 21227 G.mC. A.mC.mC.mU.mU.mU.mC.mU. A* 5  7(3) 2 mG*mA-TEG-Chl P.mU.fC.fU.A.G.mA.A.mA. G.G.fU.G.fC*mA*mA*mA*fC*mA*U CTGF L3 20392 13 2275 21381 G.mU. G. A. mC.mC. A. A. A. A. G*mU*mA-TEG- 6  6(9) 1 Chl P.mU.A.fC.fU.fU.fU.fU.G.G.fU.mC.A.mC*A* mC*mU*mC*mU*C 21383 mG*mU*mG.mA.mC.mC.mA.mA.mA.mA.mG*mU*mA-TEG- 6  6(0) 2 Chl P.mU.A.fC.fU.fU.fU.fU.G.G.fU.mC. A.mC*A*mC*mU*mC*mU*C CTGF L4 17387 5 2299 21224 mC.mC.mU.mU.mU.mC.mU. A. G.mU.mU* mG*mA- 5  9(2) 1 TEG-Chl P.mU.fC. A. A.fC.fU. A. G. A. mA. A. G. G* fU*mG*fC*mA*mA* A Table 24: Lead 21212 corresponds to SEQ ID NOs 3445 and 3446; lead 21214 G-8.D1) corresponds to SEQ ID NOs 3449 and 3450 (an unmodified form of SEQ ID NO:3450 corresponds to SEQ ID NO:4205:UCAACUAGAAAGGUGCAAA); lead 21215 corresponds to SEQ ID NOs 3451 and 3452 (an unmodified form of SEQ ID NO:3452 corresponds to SEQ ID NO:4204:UUAGAAAGGUGCAAACAAGG); lead 21204 corresponds to SEQ ID NOs 3429 and 3430; lead 21205 corresponds to SEQ ID NOs 3431 and 3432; lead 21227 corresponds to SEQ ID NOs 3475 and 3476; lead 21381 corresponds to SEQ ID NOs 3493 and 3494; lead 21383 corresponds to SEQ ID NOs 3497 and 3498; and lead 21224 corresponds to SEQ ID NOs 3469 and 3470.

TABLE 25 Summary of PTGS2 Leads Optimized Opt. Opt. Generic Targeting lead lead lead Name TEG ID 2′F site sequence Optimized lead sequence 2′F 2′OH Priority PTGS2 L1 20394 8 448 21228 G. A.mU.mC. A.mC. A.mU.mU.mU. G*mA*mA-TEG-Chl 8  6(5) 1 P.mU.fU.fC.A.mA.A.fU.G.fU.G.A.fU.fC*fU*mG*mG* mA*fU* G 21229 G. A.mU.mC. A.mC. A.mU.mU.mU. G*mA*mA-TEG-Chl 4 10(5) 3 P.mU.fU.fC.A.A.A.fU.G.fU.G.A.mU.mC*mU*G*G*A* mU*G 21230 G. A.mU.mC. A.mC. A.mU.mU.mU. G*mA*mA-TEG-Chl 8  7(5) 2 P.mU.fU.fC.A.A.A.fU.G.fU.G.A.fU.fC*fU*mG*mG* mA*fU* G PTGS2 L2 20395 8 449 21293 G. A.mU.mC. A.mC. A.mU.mU.mU. G. A*mU*mA-TEG- 5  8(6) 4 Chl P.mU. A.fU.fC. A. A. A.fU. G.fU. G. A.mU. mC*mU*mG*mG*mA*fU* G 21394 G. A.mU.mC. A.mC. A.mU.mU.mU. G. A*mU*mA-TEG- 6 11(6) 3 Chl P.mU. A.fU.fC. A. A. A.fU. G.fU. G. A.mU. fC*mU* G* G* A *fU* G 21233 G. A.mU.mC. A.mC. A.mU.mU.mU. G. A*mU*mA-TEG- 8 11(6) 1 Chl P.mU.A.fU.fC.A.A.A.fU.G.fU.G.A.fU. fC*fU*G*G*A*fU*G 21234 G. A.mU.mC. A.mC. A.mU.mU.mU. G. A*mU*mA-TEG- 7  8(6) 2 Chl P.mU.A.fU.fC.A.A.A.fU.G.fU.G.A.fU.fC* fU*mG*mG*mA*fU*G Table 25: Lead 21228 corresponds to SEQ ID NOs 4309 and 4310; lead 21229 corresponds to SEQ ID NOs 4311 and 4312; lead 21230 corresponds to SEQ ID NOs 4313 and 4314; lead 21293 corresponds to SEQ ID NOs 4315 and 4316; lead 21394 corresponds to SEQ ID NOs 4317 and 4318; lead 21233 corresponds to SEQ ID NOs 4319 and 501; and lead 21234 corresponds to SEQ ID NOs 502 and 1059.

TABLE 26 Summary of hTGFB1 Leads Optimized Opt. Generic Targeting lead Opt. lead Name site sequence Optimized lead sequence lead 2′F 2′OH Priority TGFb1 hL3 1244 21374 mC*mA*mC.mA.mC.mA.mG.mC.mA.mU.mA*mU*mA-TEG-Chl 9 6(0) 1 P.mU.A.fU.A.fU.G.fC.fU.G.fU.G.fU.mG*fU*mA*fC*mU*fC*U Table 26: lead 21374 corresponds to SEQ ID NOs 3793 and 3794.

TABLE 27 Summary of hTGFB2 Leads Optimized Opt. Generic Targeting lead Opt. lead Name site sequence Optimized lead sequence lead 2′F 2′OH Priority TGFb2 hL3 2081 21379 mU*mC*mA.mU.mC.mA.mG.mU.mG.mU.mU*mA-TEG-Chl 7 9(0) 1 P.mU.fU. A. A.fC. A.fC.fU. G. A.fU. G. A* A*fC*fC* mA*mA* G mU*mC*mA.mU.mC.mA.mG.mU.mG.mU.mU*mA*mA-TEG-Chl 7 7(0) 2 P.mU.fU. A. A.fC. A.fC.fU. G. A.fU. G.mA*mA*fC*fC* mA*mA* G Table 27: lead 21379 corresponds to SEQ ID NOs 3637, 3638, 3639 and 3640.

Having thus described several aspects of at least one embodiment of this invention, it is to be appreciated various alterations, modifications, and improvements will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of this disclosure, and are intended to be within the spirit and scope of the invention. Accordingly, the foregoing description and drawings are by way of example only.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

All references, including patent documents, disclosed herein are incorporated by reference in their entirety. This application incorporates by reference the entire contents, including all the drawings and all parts of the specification (including sequence listing or amino acid/polynucleotide sequences) of PCT Publication No. WO2010/033247 (Application No. PCT/US2009/005247), filed on Sep. 22, 2009, and entitled “REDUCED SIZE SELF-DELIVERING RNAI COMPOUNDS” and PCT Publication No. WO2009/102427 (Application No. PCT/US2009/000852), filed on Feb. 11, 2009, and entitled, “MODIFIED RNAI POLYNUCLEOTIDES AND USES THEREOF.” 

What is claimed is:
 1. A double-stranded ribonucleic acid (dsRNA) comprising a sense strand and an antisense strand, wherein the sense and/or antisense strand comprises at least 12 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 4309 (G. A.mU.mC. A.mC. A.mU.mU.mU. G*mA*mA-TEG-Chl), 4310 (P.mU.fU.fC.A.mA.A.fU.G.fU.G.A.fU.fC*fU*mG*mG*mA*fU* G), 4311 (G. A.mU.mC. A.mC. A.mU.mU.mU. G*mA*mA-TEG-Chl), 4312 (P.mU.fU.fC.A.A.A.fU.G.fU.G.A.mU.mC*mU*G*G*A*mU*G), 4313 (G. A.mU.mC. A.mC. A.mU.mU.mU. G*mA*mA-TEG-Chl), 4314 (P.mU.fU.fC.A.A.A.fU.G.fU.G.A.fU.fC*fU*mG*mG*mA*fU* G), 4315 (G. A.mU.mC. A.mC. A.mU.mU.mU. G. A*mU*mA-TEG-Chl), 4316 (P.mU. A.fU.fC. A. A. A.fU. G.fU. G. A.mU.mC*mU*mG*mG*mA*fU* G), 4317 (G. A.mU.mC. A.mC. A.mU.mU.mU. G. A*mU*mA-TEG-Chl), 4318 (P.mU. A.fU.fC. A. A. A.fU. G.fU. G. A.mU.fC*mU* G* G* A*fU* G), 4319 (G. A.mU.mC. A.mC. A.mU.mU.mU. G. A*mU*mA-TEG-Chl), 501 (P.mU.A.fU.fC.A.A.A.fU.G.fU.G.A.fU.fC*fU*G*G*A*fU*G), 502 (G. A.mU.mC. A.mC. A.mU.mU.mU. G. A*mU*mA-TEG-Chl), and 1059 (P.mU.A.fU.fC.A.A.A.fU.G.fU.G.A.fU.fC*fU*mG*mG*mA*fU*G), wherein the dsRNA is an sd-rxRNA, wherein the antisense strand is 16-23 nucleotides long and the sense strand is 8-15 nucleotides long, wherein the sd-rxRNA includes a double-stranded region and a single-stranded region, wherein the double-stranded region is from 8-15 nucleotides long, wherein the single-stranded region is at the 3′ end of the antisense strand and is 4-12 nucleotides long, wherein the single-stranded region contains 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 phosphorothioate modifications, and wherein at least 40% of the nucleotides of the double-stranded nucleic acid molecule are modified.
 2. The dsRNA of claim 1, wherein the dsRNA is hydrophobically modified or wherein the dsRNA is linked to a hydrophobic conjugate.
 3. A composition comprising the dsRNA of claim
 1. 4. The composition of claim 3 wherein the composition is (i) formulated for delivery to the skin; (ii) in a neutral formulation; (iii) formulated for topical delivery; or (iv) formulated for intradermal injection.
 5. A method comprising administering the dsRNA of claim 1 to the skin of a subject in need thereof.
 6. The method of claim 5, wherein the method is a method for treating or preventing a fibrotic disorder.
 7. The method of claim 6, wherein the fibrotic disorder is selected from the group consisting of pulmonary fibrosis, liver cirrhosis, scleroderma and glomerulonephritis, liver fibrosis, skin fibrosis, muscle fibrosis, radiation fibrosis, kidney fibrosis, proliferative vitreoretinopathy, restenosis and uterine fibrosis, and scarring resulting in the failure of a trabeculectomy. 